WO2003104397A2 - Modulation antisens de l'expression de la proteine kinase gr 6 - Google Patents

Modulation antisens de l'expression de la proteine kinase gr 6 Download PDF

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WO2003104397A2
WO2003104397A2 PCT/US2003/017174 US0317174W WO03104397A2 WO 2003104397 A2 WO2003104397 A2 WO 2003104397A2 US 0317174 W US0317174 W US 0317174W WO 03104397 A2 WO03104397 A2 WO 03104397A2
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protein
acid
receptor kinase
compound
coupled receptor
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WO2003104397A3 (fr
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Susan M. Freier
Kenneth W. Dobie
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Isis Pharmaceuticals Inc.
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
    • A—HUMAN NECESSITIES
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    • C12N2310/00—Structure or type of the nucleic acid
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/321—2'-O-R Modification
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    • C12N2310/30—Chemical structure
    • C12N2310/33—Chemical structure of the base
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    • C12N2310/3341—5-Methylcytosine
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    • C12N2310/00—Structure or type of the nucleic acid
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    • C12N2310/34—Spatial arrangement of the modifications
    • C12N2310/346—Spatial arrangement of the modifications having a combination of backbone and sugar modifications
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
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Definitions

  • the present invention provides compositions and methods for modulating the expression of G protein-coupled receptor kinase 6.
  • this invention relates to compounds, particularly oligonucleotides, specifically hybridizable with nucleic acids encoding G protein-coupled receptor kinase 6. Such compounds have been shown to modulate the expression of G protein-coupled receptor kinase 6.
  • G protein-coupled receptors are 7-transmembrane domain-containing proteins activated by a wide variety of agonists (hormones, neurotransmitters, opioids, odorants, light) and these receptors serve to transduce the ligand- binding or activating event into an intracellular signal.
  • GPCRs Upon agonist stimulation, GPCRs catalyze nucleotide exchange (GDP to GTP) and thereby activate intracellular G-proteins, and the GPCR must be turned off to prevent continuing G-protein activation.
  • G protein-coupled receptor kinases are primarily responsible for the rapid loss of GPCR responsiveness in spite of continuous presence of the agonist, in a process known as homologous desensitization.
  • Phosphorylation of activated GPCRs by GRKs and by other second messenger-activated receptor-specific protein kinases, such as protein kinase C (PKC) leads to a shortened physiologic response.
  • PKC protein kinase C
  • Activated receptor phosphorylation promotes binding of regulatory arrestin proteins, which prevent further receptor-coupling to G-proteins, thereby impeding additional rounds of receptor-activated signal transduction (Palczewski, Eur. J. Biochem . , 1997, 248, 261-269).
  • the first comprises the closely related GRK2 and GRK3
  • the second includes GRK1 , GRK4 , GRK5 and GRK6.
  • the amino terminal domains of all GRKs are highly similar in sequence and is believed to be involved in the interaction with activated GPCRs, and, in GRK4 , GRK5 and GRK6 , may also be involved in binding phosphatidylinositol 4,5 bisphosphate (Palczewski, Eur. J. Biochem . , 1997, 248 , 261-269).
  • a human G protein-coupled receptor kinase 6 cDNA clone was isolated.
  • Two major RNA species of approximately 2.4- and 3-kilobases were observed by Northern blot analysis .
  • the larger transcript appeared to be expressed at comparable levels in brain, skeletal muscle and pancreas, with lower levels found in lung, placenta, heart and kidney.
  • the smaller transcript was found predominantly in skeletal muscle with lower levels in brain and pancreas (Benovic and Gomez, J. Biol . Chem.
  • G protein-coupled receptor kinase 6 was mapped to chromosomal region 5q35 near a cluster of growth factor and neurotransmitter receptors (Bullrich et al . , Cytogrenet. Cell Genet . , 1995, 70, 250-254).
  • a pseudogene related to G protein-coupled receptor kinase 6 was also identified and mapped to a distinct location on chromosome 13 (Bullrich et al . , Cytogrenet. Cell Genet . , 1995, 70, 250-254; Gagnon and Benovic, Gene, 1991 , 184 , 13-19; Haribabu and Snyderman, Proc . Natl . Acad . Sci . U. S . A . , 1993, 90 , 9398- 9402) .
  • the rat and mouse homologues of G protein-coupled receptor kinase 6 have also been cloned (Fehr et al . , Brain Res . Brain Res . Protoc , 1999, 3, 242-251; Neill et al . , Endocrinology, 1996, 137, 3942-3947) and four distinct mouse isoforms, mGRK6-A through mGRK6-D, generated by alternative splicing, have been identified (Moepps et al . , Genomics, 1999, 60, 199-209) .
  • G protein-coupled receptor kinase 6 increases its activity both by increasing its hydrophobicity and localizing it near its membrane-bound substrates, and by increasing its kinase catalytic activity (Stoffel et al . , Biochemistry, 1998, 37, 16053-16059; Stoffel et al . , J " . Biol . Chem . , 1994, 269, 211'91-27 '94) .
  • G protein-coupled receptor kinase 6 phosphorylates and desensitizes a number of agonist-activated G protein-coupled receptors.
  • Rhodopsin and the ⁇ 2 -adrenergic and m2 muscarinic cholinergic receptors are stimulus-dependent substrates for G protein-coupled receptor kinase 6 (Loudon and Benovic, J.
  • G protein-coupled receptor kinase 6 increases the extent of ⁇ -opioid receptor desensitization upon agonist stimulation, suggesting a potential role for G protein-coupled receptor kinase 6 in drug tolerance (Willets and Kelly, Eur. J. Pharmacol . , 2001, 431 , 133-141).
  • G protein-coupled receptor kinase 6 protein levels are upregulated in rat brains in response to chronic treatment with the ⁇ -opioid receptor agonist sufentanil, further implicating G proteincoupled receptor kinase 6 in the short- and long-term adaptive changes in ⁇ -opioid receptor activity that contribute to development of analgesic tolerance in living animals (Hurle, J. Neurochem . , 2001, 77, 486-492).
  • the activity of G protein-coupled receptor kinase 6 may be modulated by phosphorylation and is inhibited by calmodulin and calcium.
  • G protein-coupled receptor kinase 6 possesses a putative autophosphorylation motif and although it is poorly autophosphorylated in vi tro, the protein has been shown to undergo autophosphorylation in vivo in COS-7 cells (Milcent et al., Biochem . Biophys . Res . Commun . , 1999, 259, 224-229).
  • Ca2+/calmodulin acts as a potent inhibitor of G protein- coupled receptor kinase 6 activity (Pronin et al . , J " . Biol . Chem . , 1991 , 272 , 18273-18280).
  • Impairment in ⁇ -adrenergic receptor activation and alterations in vascular responsiveness to ⁇ -adrenergic agonists is believed to play an important role in development and/or maintenance of hypertension.
  • GRK activity is increased in human lymphocytes from hypertensive subjects, and while this has not been attributed to altered activity of G proteincoupled receptor kinase 6 specifically, a possible role for G protein-coupled receptor kinase 6 in hypertension has also not been conclusively ruled out (Gros et al . , J. Clin . Invest . , 1997 , 99, 2087-2093) .
  • G protein-coupled receptor kinase 6 is expressed at a much higher level in pregnant term myometrium than in non-pregnant uterine tissue, and it has been suggested that G protein-coupled receptor kinase 6 may regulate uterine contractility at term (Brenninkmeijer et al . , J " . Endocrinol . , 1999, 162, 401-408).
  • G protein-coupled receptor kinase 6 appears to play an important role in modulating GPCR function in immune cells (Loudon et al . , Blood, 1996 , 88 , 4547-4557) .
  • Proinflammatory chemokines and cytokines and their receptors are known to play an important role in chronic inflammatory diseases by initiating and maintaining the local inflammatory process through recruitment of monocytes and lymphocytes .
  • the mAb A16/17 functionally inhibits G protein-coupled receptor kinase activity, reducing agonist-induced phosphorylation of the receptor (Oppermann et al., Proc . Natl . Acad . Sci . U. S . A . , 1996, 93 , 7649-7654) .
  • the same mAb was used to block G protein-coupled receptor kinase activity (measured as the level of phosphorylation of rhodopsin) in membrane extracts of late pregnant rat myometrium, demonstrating that ⁇ -adrenergic signaling at parturition may be triggered by GRKs (Simon et al . , Endocrinology, 2001, 142 , 1899-1905) .
  • G protein-coupled receptor kinase 6 can phosphorylate the rat follicle stimulating hormone receptor (rFSHR) , and a kinase-deficient dominant negative mutant GRK6- (K215M, K216M) effectively inhibits agonist-induced phosphorylation of the rFSHR (Lazari et al . , Mol . Endocrinol . , 1999, 13 , 866-878).
  • the thromboxane A 2 receptor mediates vasoconstriction, mitogenesis, and platelet aggregation, and has been shown to undergo rapid agonist-induced desensitization.
  • GRKs The role of GRKs in the phosphorylation and desensitization of the alpha isoform of this receptor was investigated, and it was discovered that a known PKC inhibitor, GF 109203X, also inhibited GRKs, including G protein-coupled receptor kinase 6. Because GF 109203X acts as an inhibitor of multiple kinases, it has been suggested that this compound may interact with the nucleotide-binding site common to all kinases, but the mechanism by which it inhibits G protein coupled-receptor kinase 6 remains to be elucidated (Zhou et al . , J " . Pharmacol . Exp . Ther. , 2001, 298, 1243-1251).
  • a phosphorothioate antisense oligonucleotide, 17 nucleotides in length and targeted to the initiation codon of the G protein coupled-receptor kinase 6 mRNA was used to investigate the role of G protein coupled-receptor kinase 6 in desensitization of histamine H 2 receptors in the human gastric carcinoma cell line MKN-45, and it was concluded that ⁇ - adrenergic receptor kinase 1 but not G protein coupled- receptor kinase 6, is involved in desensitization of H 2 receptors (Nakata et al . , Digestion, 1996, 57, 406-410).
  • 5,591,618 is a purified and isolated polynucleotide that is a genomic DNA, a cDNA, a plasmid insert DNA, or a wholly or partially chemically synthesized DNA, encoding a mammalian G protein-coupled receptor kinase 6 (GRK6) enzyme, a host cell stably transformed or transfected with said DNA, a method for producing GRK6 enzyme, a purified and isolated mammalian GRK6 enzyme, a mammalian GRK6 enzyme encoded by a full length DNA sequence which hybridizes to the non-coding strand corresponding to the DNA representing the coding sequence of the G protein-coupled receptor kinase 6 gene.
  • Antisense oligonucleotides are generally disclosed (Chantry et al . , 1997; Chantry et al . , 1996).
  • US Patent 6,255,069 Disclosed and claimed in US Patent 6,255,069 is an essentially pure cDNA encoding wild-type GRK5 or GRK6, dominant negative mutations of wild-type GRK5 or GRK6 and fragments thereof for use as probes, a recombinant DNA expression vector containing said cDNA, a process for preparing an essentially pure G protein-coupled kinase, and a cell line comprising Sf9 insect cells capable of expressing a G protein-coupled receptor kinase comprising wild-type GRK5 or GRK6, dominant negative mutations of wild-type GRK5 or GRK6 and fragments thereof for use as probes.
  • Antisense oligonucleotides are generally disclosed (Benovic et al . , 2001) .
  • a peptide comprising a peptide derivative of the ⁇ D region of a protein kinase, wherein said protein has between about five and about thirty amino acids or amino acid analogs and said peptide modulates activity of the protein kinase, and wherein the protein kinase is a member of a protein kinase family selected from a group of which the G protein-coupled receptor kinases and specifically G protein-coupled receptor kinase 6 are members.
  • a method of identifying a peptide which modulates the activity of a protein kinase a method of modulating the activity of a protein kinase in a subject, a method of detecting a ligand that binds to the ⁇ D region of a protein kinase, and an antibody as well as a method of producing antibodies that immunologically bind to the ⁇ D region of a protein kinase (Ben-Sasson, 2000) .
  • Antisense technology is emerging as an effective means for reducing the expression of specific gene products and may therefore prove to be uniquely useful in a number of therapeutic, diagnostic, and research applications for the modulation of G protein-coupled receptor kinase 6 expression.
  • the present invention provides compositions and methods for modulating G protein-coupled receptor kinase 6 expression.
  • the present invention is directed to compounds, particularly antisense oligonucleotides, which are targeted to a nucleic acid encoding G protein-coupled receptor kinase 6, and which modulate the expression of G protein-coupled receptor kinase 6.
  • Pharmaceutical and other compositions comprising the compounds of the invention are also provided. Further provided are methods of modulating the expression of G protein-coupled receptor kinase 6 in cells or tissues comprising contacting said cells or tissues with one or more of the antisense compounds or compositions of the invention.
  • the present invention employs oligomeric compounds, particularly antisense oligonucleotides, for use in modulating the function of nucleic acid molecules encoding G proteincoupled receptor kinase 6, ultimately modulating the amount of G protein-coupled receptor kinase 6 produced. This is accomplished by providing antisense compounds which specifically hybridize with one or more nucleic acids encoding G protein-coupled receptor kinase 6.
  • target nucleic acid and “nucleic acid encoding G protein-coupled receptor kinase 6” encompass DNA encoding G protein-coupled receptor kinase 6, RNA (including pre-mRNA and mRNA) transcribed from such DNA, and also cDNA derived from such RNA.
  • RNA including pre-mRNA and mRNA
  • cDNA derived from such RNA.
  • the specific hybridization of an oligomeric compound with its target nucleic acid interferes with the normal function of the nucleic acid. This modulation of function of a target nucleic acid by compounds which specifically hybridize to it is generally referred to as "antisense" .
  • the functions of DNA to be interfered with include replication and transcription.
  • RNA to be interfered with include all vital functions such as, for example, translocation of the RNA to the site of protein translation, translocation of the RNA to sites within the cell which are distant from the site of RNA synthesis, translation of protein from the RNA, splicing of the RNA to yield one or more mRNA species, and catalytic activity which may be engaged in or facilitated by the RNA.
  • the overall effect of such interference with target nucleic acid function is modulation of the expression of G protein-coupled receptor kinase 6.
  • modulation means either an increase (stimulation) or a decrease (inhibition) in the expression of a gene.
  • inhibition is the preferred form of modulation of gene expression and mRNA is a preferred target.
  • Targeting an antisense compound to a particular nucleic acid is a multistep process. The process usually begins with the identification of a nucleic acid sequence whose function is to be modulated. This may be, for example, a cellular gene (or mRNA transcribed from the gene) whose expression is associated with a particular disorder or disease state, or a nucleic acid molecule from an infectious agent. In the present invention, the target is a nucleic acid molecule encoding G protein- coupled receptor kinase 6.
  • the targeting process also includes determination of a site or sites within this gene for the antisense interaction to occur such that the desired effect, e.g., detection or modulation of expression of the protein, will result.
  • a preferred intragenic site is the region encompassing the translation initiation or termination codon of the open reading frame (ORF) of the gene. Since, as is known in the art, the translation initiation codon is typically 5 ' -AUG (in transcribed mRNA molecules; 5 ' -ATG in the corresponding DNA molecule) , the translation initiation codon is also referred to as the "AUG codon, " the "start codon” or the "AUG start codon” .
  • translation initiation codon having the RNA sequence 5'-GUG, 5 ' -UUG or 5'-CUG, and 5 ' -AUA, 5 ' -ACG and 5 ' -CUG have been shown to function in vivo .
  • the terms "translation initiation codon” and "start codon” can encompass many codon sequences, even though the initiator amino acid in each instance is typically methionine (in eukaryotes) or formylmethionine (in prokaryotes) .
  • start codon and “translation initiation codon” refer to the codon or codons that are used in vivo to initiate translation of an mRNA molecule transcribed from a gene encoding G protein-coupled receptor kinase 6, regardless of the sequence (s) of such codons.
  • a translation termination codon (or "stop codon”) of a gene may have one of three sequences, i.e., 5 ' -UAA, 5 ' -UAG and 5 ' -UGA (the corresponding DNA sequences are 5 ' -TAA, 5 ' -TAG and 5 ' -TGA, respectively) .
  • start codon region and “translation initiation codon region” refer to a portion of such an mRNA or gene that encompasses from about 25 to about 50 contiguous nucleotides in either direction (i.e., 5' or 3 ' ) from a translation initiation codon.
  • the terms “start codon region” and “translation initiation codon region” refer to a portion of such an mRNA or gene that encompasses from about 25 to about 50 contiguous nucleotides in either direction (i.e., 5' or 3 ' ) from a translation initiation codon.
  • stop codon region and “translation termination codon region” refer to a portion of such an mRNA or gene that encompasses from about 25 to about 50 contiguous nucleotides in either direction (i.e., 5' or 3 ' ) from a translation termination codon.
  • Other target regions include the 5 ' untranslated region
  • (5 'UTR) known in the art to refer to the portion of an mRNA in the 5' direction from the translation initiation codon, and thus including nucleotides between the 5 ' cap site and the translation initiation codon of an mRNA or corresponding nucleotides on the gene, and the 3' untranslated region
  • 3'UTR 3'UTR
  • the 5' cap of an mRNA comprises an N7-methylated guanosine residue joined to the 5 ' -most residue of the mRNA via a 5 ' -5 ' triphosphate linkage.
  • the 5' cap region of an mRNA is considered to include the 5 ' cap structure itself as well as the first 50 nucleotides adjacent to the cap. The 5' cap region may also be a preferred target region.
  • mRNA splice sites i.e., intron- exon junctions
  • intron- exon junctions may also be preferred target regions, and are particularly useful in situations where aberrant splicing is implicated in disease, or where an overproduction of a particular mRNA splice product is implicated in disease. Aberrant fusion junctions due to rearrangements or deletions are also preferred targets.
  • fusion transcripts mRNA transcripts produced via the process of splicing of two (or more) mRNAs from different gene sources are known as "fusion transcripts" . It has also been found that introns can be effective, and therefore preferred, target regions for antisense compounds targeted, for example, to DNA or pre-mRNA.
  • RNA transcripts can be produced from the same genomic region of DNA. These alternative transcripts are generally known as "variants”. More specifically, “pre-mRNA variants” are transcripts produced from the same genomic DNA that differ from other transcripts produced from the same genomic DNA in either their start or stop position and contain both intronic and extronic regions.
  • pre-mRNA variants Upon excision of one or more exon or intron regions or portions thereof during splicing, pre-mRNA variants produce smaller "mRNA variants". Consequently, mRNA variants are processed pre-mRNA variants and each unique pre-mRNA variant must always produce a unique mRNA variant as a result of splicing. These mRNA variants are also known as "alternative splice variants". If no splicing of the pre-mRNA variant occurs then the pre-mRNA variant is identical to the mRNA variant . It is also known in the art that variants can be produced through the use of alternative signals to start or stop transcription and that pre-mRNAs and mRNAs can possess more that one start codon or stop codon.
  • Alterants that originate from a pre-mRNA or mRNA that use alternative start codons are known as "alternative start variants" of that pre-mRNA or mRNA.
  • Those transcripts that use an alternative stop codon are known as “alternative stop variants” of that pre-mRNA or mRNA.
  • One specific type of alternative stop variant is the "polyA variant” in which the multiple transcripts produced result from the alternative selection of one of the "polyA stop signals" by the transcription machinery, thereby producing transcripts that terminate at unique polyA sites.
  • oligonucleotides are chosen which are sufficiently complementary to the target, i.e., hybridize sufficiently well and with sufficient specificity, to give the desired effect.
  • hybridization means hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleoside or nucleotide bases.
  • adenine and thymine are complementary nucleobases which pair through the formation of hydrogen bonds.
  • “Complementary,” as used herein, refers to the capacity for precise pairing between two nucleotides.
  • oligonucleotide and the DNA or RNA are considered to be complementary to each other at that position.
  • the oligonucleotide and the DNA or RNA are complementary to each other when a sufficient number of corresponding positions in each molecule are occupied by nucleotides which can hydrogen bond with each other.
  • “specifically hybridizable” and “complementary” are terms which are used to indicate a sufficient degree of complementarity or precise pairing such that stable and specific binding occurs between the oligonucleotide and the DNA or RNA target. It is understood in the art that the sequence of an antisense compound need not be 100% complementary to that of its target nucleic acid to be specifically hybridizable.
  • An antisense compound is specifically hybridizable when binding of the compound to the target DNA or RNA molecule interferes with the normal function of the target DNA or RNA to cause a loss of activity, and there is a sufficient degree of complementarity to avoid non-specific binding of the antisense compound to non-target sequences under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in -.vivo assays or therapeutic treatment, and in the case of in vitro assays, under conditions in which the assays are performed.
  • the antisense compounds of the present invention comprise at least 80% sequence complementarity to a target region within the target nucleic acid, moreover that they comprise 90% sequence complementarity and even more comprise 95% sequence complementarity to the target region within the target nucleic acid sequence to which they are targeted.
  • an antisense compound in which 18 of 20 nucleobases of the antisense compound are complementary, and would therefore specifically hybridize, to a target region would represent 90 percent complementarity.
  • Percent complementarity of an antisense compound with a region of a target nucleic acid can be determined routinely using basic local alignment search tools (BLAST programs) (Altschul et al . , J. Mol . Biol . , 1990, 215, 403-410; Zhang and Madden, Genome Res . , 1997, 7, 649-656) .
  • Antisense and other compounds of the invention which hybridize to the target and inhibit expression of the target, are identified through experimentation, and representative sequences of these compounds are hereinbelow identified as preferred embodiments of the invention.
  • the sites to which these preferred antisense compounds are specifically hybridizable are hereinbelow referred to as "preferred target regions" and are therefore preferred sites for targeting.
  • preferred target region is defined as at least an 8-nucleobase portion of a target region to which an active antisense compound is targeted. While not wishing to be bound by theory, it is presently believed that these target regions represent regions of the target nucleic acid- which are accessible for hybridization.
  • Target regions 8-80 nucleobases in length comprising a stretch of at least eight (8) consecutive nucleobases selected from within the illustrative preferred target regions are considered to be suitable preferred target regions as well .
  • Exemplary good preferred target regions include DNA or RNA sequences that comprise at least the 8 consecutive nucleobases from the 5' -terminus of one of the illustrative preferred target regions (the remaining nucleobases being a consecutive stretch of the same DNA or RNA beginning immediately upstream of the 5' -terminus of the target region and continuing until the DNA or RNA contains about 8 to about
  • good preferred target regions are represented by DNA or RNA sequences that comprise at least the 8 consecutive nucleobases from the 3' -terminus of one of the illustrative preferred target regions (the remaining nucleobases being a consecutive stretch of the same DNA or RNA beginning immediately downstream of the 3' -terminus of the target region and continuing until the DNA or RNA contains about 8 to about 80 nucleobases) .
  • One having skill in the art once armed with the empirically-derived preferred target regions illustrated herein will be able, without undue experimentation, to identify further preferred target regions.
  • one having ordinary skill in the art will also be able to identify additional compounds, including oligonucleotide probes and primers, that specifically hybridize to these preferred target regions using techniques available to the ordinary practitioner in the art .
  • Antisense compounds are commonly used as research reagents and diagnostics. For example, antisense oligonucleotides, which are able to inhibit gene expression with 17, specificity, are often used by those of ordinary skill to elucidate the function of particular genes. Antisense compounds are also used, for example, to distinguish between functions of various members of a biological pathway.
  • Antisense modulation has, therefore, been harnessed for research use.
  • the antisense compounds of the present invention can be used as tools in differential and/or combinatorial analyses to elucidate expression patterns of a portion or the entire complement of genes expressed within cells and tissues.
  • Expression patterns within cells or tissues treated with one or more antisense compounds are compared to control cells or tissues not treated with antisense compounds and the patterns produced are analyzed for differential levels of gene expression as they pertain, for example, to disease association, signaling pathway, cellular localization-, expression level, size, structure or function of the genes examined. These analyses can be performed on stimulated or unstimulated cells and in the presence or absence of other compounds which affect expression patterns .
  • Examples of methods of gene expression analysis known in the art include DNA arrays or microarrays (Brazma and Vilo, FEBS Lett . , 2000, 480, 17-24; Celis, et al . , FEBS Lett . , 2000, 480, 2-16) , SAGE (serial analysis of gene expression) (Madden, et al . , Drug Discov. Today, 2000, 5, 415-425), READS
  • Antisense oligonucleotides have been employed as therapeutic moieties in the treatment of disease states in animals and man.
  • Antisense oligonucleotide drugs, including ribozymes, have been safely and effectively administered to humans and numerous clinical trials are presently underway. It is thus established that oligonucleotides can be useful therapeutic modalities that can be configured to be useful in treatment regimes for treatment of cells, tissues and animals, especially humans.
  • oligonucleotide refers to an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or mimetics thereof.
  • RNA ribonucleic acid
  • DNA deoxyribonucleic acid
  • oligonucleotides composed of naturally-occurring nucleobases, sugars and covalent internucleoside (backbone) linkages as well as oligonucleotides having non-naturally-occurring portions which function similarly.
  • Such modified or substituted oligonucleotides are often preferred over native forms because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for nucleic acid target and increased stability in the presence of nucleases .
  • antisense oligonucleotides are a preferred form of antisense compound
  • the present invention comprehends other oligomeric antisense compounds, including but not limited to oligonucleotide mimetics such as are described below.
  • the antisense compounds in accordance with this invention preferably comprise from about 8 to about 80 nucleobases (i.e. from about 8 to about 80 linked nucleosides) .
  • Particularly preferred antisense compounds are antisense oligonucleotides from about 8 to about 50 nucleobases, even more preferably those comprising from about 12 to about 30 nucleobases.
  • Antisense compounds include ribozymes, external guide sequence (EGS) oligonucleotides
  • Antisense compounds 8-80 nucleobases in length comprising a stretch of at least eight (8) consecutive nucleobases selected from within the illustrative antisense compounds are considered to be suitable antisense compounds as well .
  • Exemplary preferred antisense compounds include DNA or RNA sequences that comprise at least the 8 consecutive nucleobases from the 5' -terminus of one of the illustrative preferred antisense compounds (the remaining nucleobases being a consecutive stretch of the same DNA or RNA beginning immediately upstream of the 5' -terminus of the antisense compound which is specifically hybridizable to the target nucleic acid and continuing until the DNA or RNA contains about 8 to about 80 nucleobases) .
  • preferred antisense compounds are represented by DNA or RNA sequences that comprise at least the 8 consecutive nucleobases from the 3' -terminus of one of the illustrative preferred antisense compounds (the remaining nucleobases being a consecutive stretch of the same DNA or RNA beginning immediately downstream of the 3' -terminus of the antisense compound which is specifically hybridizable to the target nucleic acid and continuing until the DNA or RNA contains about 8 to about 80 nucleobases) .
  • One having skill in the art once armed with the empirically-derived preferred antisense compounds illustrated herein will be able, without undue experimentation, to identify further preferred antisense compounds .
  • Antisense and other compounds of the invention which hybridize to the target and inhibit expression of the target, are identified through experimentation, and representative sequences of these compounds are herein identified as preferred embodiments of the invention. While specific sequences of the antisense compounds are set forth herein, one of skill in the art will recognize that these serve to illustrate and describe particular embodiments within the scope of the present invention. Additional preferred antisense compounds may be identified by one having ordinary skill.
  • a nucleoside is a base-sugar combination. The base portion of the nucleoside is normally a heterocyclic base. The two most common classes of such heterocyclic bases are the purines and the pyrimidines .
  • Nucleotides are nucleosides that further include a phosphate group covalently linked to the sugar portion of the nucleoside.
  • the phosphate group can be linked to either the 2', 3' or 5' hydroxyl moiety of the sugar.
  • the phosphate groups covalently link adjacent nucleosides to one another to form a linear polymeric compound.
  • the respective ends of this linear polymeric structure can be further joined to form a circular structure, however, open linear structures are generally preferred.
  • linear structures may also have internal nucleobase complementarity and may therefore fold in a manner as to produce a double stranded structure.
  • the phosphate groups are commonly referred to as forming the internucleoside backbone of the oligonucleotide.
  • the normal linkage or backbone of RNA and DNA is a 3' to 5' phosphodiester linkage.
  • oligonucleotides containing modified backbones or non-natural internucleoside linkages include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone.
  • modified oligonucleotides that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides .
  • Preferred modified oligonucleotide backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotri- esters, methyl and other alkyl phosphonates including 3' - alkylene phosphonates, 5 ' -alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3'- amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphates and borano- phosphates having normal 3' -5' linkages, 2' -5' linked analogs of these, and those having inverted polarity wherein one or more internucleotide linkages is a 3 ' to 3 ' , 5' to 5 ' or 2 ' to 2'
  • Preferred oligonucleotides having inverted polarity comprise a single 3' to 3 ' linkage at the 3 ' -most internucleotide linkage i.e. a single inverted nucleoside residue which may be abasic (the nucleobase is missing or has a hydroxyl group in place thereof) .
  • Various salts, mixed salts and free acid forms are also included.
  • Preferred modified oligonucleotide backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages.
  • morpholino linkages formed in part from the sugar portion of a nucleoside
  • siloxane backbones sulfide, sulfoxide and sulfone backbones
  • formacetyl and thioformacetyl backbones methylene formacetyl and thioformacetyl backbones
  • riboacetyl backbones alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH 2 component parts.
  • oligonucleosides include, but are not limited to, U.S.: 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938, 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307, 5,561,225; 5,596,086; 5,602,240; 5,610,289; 5,602,240 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312
  • both the sugar and the internucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups.
  • the base units are maintained for hybridization with an appropriate nucleic acid target compound.
  • an oligomeric compound an oligonucleotide mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA) .
  • PNA peptide nucleic acid
  • the sugar- backbone of an oligonucleotide is replaced with an amide containing backbone, in particular an aminoethylglycine backbone.
  • nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone.
  • Representative United States patents that teach the preparation of PNA compounds include, but are not limited to, U.S.: 5,539,082; 5,714,331; and 5,719,262, each of which is herein incorporated by reference. Further teaching of PNA compounds can be found in Nielsen et al . , Science, 1991, 254, 1497-1500.
  • Most preferred embodiments of the invention are oligonucleotides with phosphorothioate backbones and oligonucleosides with heteroatom backbones, and in particular -CH2-NH-O-CH2-, -CH 2 -N(CH 3 ) -O-CH2- [known as a methylene (methylimino) or MMI backbone] , -CH 2 -0-N (CH 3 ) -CH 2 - , -CH 2 -N(CH 3 )- N(CH 3 )-CH 2 - and -O-N (CH 3 ) -CH 2 -CH 2 - [wherein the native phosphodiester backbone is represented as -0-P-O-CH 2 -] of the above referenced U.S.
  • Preferred oligonucleotides comprise one of the following at the 2' position: OH; F; 0-, S-, or N-alkyl; 0-, S-, or N-alkenyl; O- , S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted Ci to C ⁇ 0 alkyl or C 2 to C ⁇ 0 alkenyl and alkynyl.
  • oligonucleotides comprise one of the following at the 2' position: Ci to Cio lower alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl, 0-alkaryl or O- aralkyl, SH, SCH 3 , OCN, Cl , Br, CN, CF 3 , OCF 3 , S0CH 3 , S0 2 CH 3 , 0N0 2 , N0 2 , N 3 , NH 2 , heterocycloalkyl, heterocycloalkaryl , aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents having similar properties.
  • a preferred modification includes 2 ' -methoxyethoxy (2 ' -0-CH 2 CH 2 0CH 3 , also known as 2'-0-(2- methoxyethyl) or 2 ' -MOE) (Martin et al . , Helv. Chim. Acta, 1995, 78, 486-504) i.e., an alkoxyalkoxy group.
  • a further preferred modification includes 2 ' -dimethylaminooxyethoxy, i.e., a 0 (CH 2 ) 2 ON(CH 3 ) 2 group, also known as 2'-DMA0E, as described in examples hereinbelow, and 2 ' -dimethylamino- ethoxyethoxy (also known in the art as 2 ' -O-dimethyl-amino- ethoxy-ethyl or 2 ' -DMAEOE) , i.e., 2 ⁇ -0-CH 2 -0-CH 2 -N (CH 3 ) 2 , also described in examples hereinbelow.
  • 2 ' -dimethylaminooxyethoxy i.e., a 0 (CH 2 ) 2 ON(CH 3 ) 2 group, also known as 2'-DMA0E, as described in examples hereinbelow
  • 2 ' -dimethylamino- ethoxyethoxy also known in the
  • Oligonucleotides may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar.
  • a further preferred modification includes Locked Nucleic Acids (LNAs) in which the 2 ' -hydroxyl group is linked to the 3 ' or 4 ' carbon atom of the sugar ring thereby forming a bicyclic sugar moiety.
  • the linkage is preferably a methelyne (-CH 2 -) n group bridging the 2' oxygen atom and the 4' carbon atom wherein n is 1 or 2.
  • LNAs and preparation thereof are described in WO 98/39352 and WO 99/14226.
  • Oligonucleotides may also include nucleobase (often referred to in the art simply as "base”) modifications or substitutions.
  • unmodified or “natural” nucleobases include the purine bases adenine (A) and guanine (G) , and the pyrimidine bases thymine (T) , cytosine (C) and uracil (U) .
  • Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C) , 5- hydroxymethyl cytosine, xanthine, hypoxanthine , 2- aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2 -propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine,
  • 5-halouracil and cytosine 5-propynyl (-C ⁇ C-CH 3 ) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6- azo uracil, cytosine and thymine, 5-uracil (pseudouracil) , 4- thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5- substituted uracils and cytosines, 7-methylguanine and 7- methyladenine, 2-F-adenine, 2 -amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3- deazaguanine and 3-deazaadenine .
  • nucleobases include tricyclic pyrimidines such as phenoxazine cytidine(lH- pyrimido [5,4-b] [1, 4] benzoxazin-2 (3H) -one) , phenothiazine cytidine (lH-pyrimido [5, 4-b] [1, 4] benzothiazin-2 (3H) -one) , G- clamps such as a substituted phenoxazine cytidine (e.g.
  • Modified nucleobases may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deaza-adenine, 7- deazaguanosine, 2-aminopyridine and 2-pyridone.
  • Further nucleobases include those disclosed in United States Patent No. 3,687,808, those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J.I., ed. John Wiley & Sons, 1990, those disclosed by Englisch et al . , Angewandte Chemie, International Edition, 1991, 30 , 613, and those disclosed by Sanghvi, Y.S., Chapter 15, Antisense Research and Applications, pages 289-302, Crooke,
  • nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds of the invention.
  • These include 5 -substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyl- adenine, 5-propynyluracil and 5-propynylcytosine .
  • 5- methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2°C (Sanghvi, Y.S., Crooke, S.T.
  • oligonucleotides of the invention involves chemically linking to the oligonucleotide one or more moieties or conjugates which enhance the activity, cellular distribution or cellular uptake of the oligonucleotide.
  • the compounds of the invention can include conjugate groups covalently bound to functional groups such as primary or secondary hydroxyl groups.
  • Conjugate groups of the invention include intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, polyethers, groups that enhance the pharmacodynamic properties of oligomers, and groups that enhance the pharmacokinetic properties of oligomers.
  • Typical conjugate groups include cholesterols, lipids, phospholipids, biotin, phenazine, folate, phenanthridine , anthraquinone , acridine, fluoresceins, rhodamines, coumarins, and dyes.
  • Groups that enhance the pharmacodynamic properties include groups that improve oligomer uptake, enhance oligomer resistance to degradation, and/or strengthen sequence-specific hybridization with RNA.
  • Groups that enhance the pharmacokinetic properties include groups that improve oligomer uptake, distribution, metabolism or excretion.
  • Conjugate moieties include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger et al . , Proc. Natl. Acad. Sci. USA, 1989, 86, 6553- 6556), cholic acid (Manoharan et al., Bioorg . Med. Chem. Let., 1994, 4, 1053-1060), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad.
  • lipid moieties such as a cholesterol moiety (Letsinger et al . , Proc. Natl. Acad. Sci. USA, 1989, 86, 6553- 6556), cholic acid (Manoharan et al., Bioorg . Med. Chem. Let., 1994, 4, 1053-1060), a thioether, e.g., hexyl-S-
  • a phospholipid e.g., di-hexadecyl-rac-glycerol or triethyl- ammonium 1, 2-di-0-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al . , Nucl. Acids Res., 1990, 18, 3777-3783) , a polyamine or a polyethylene glycol chain (Manoharan et al .
  • Oligonucleotides of the invention may also be conjugated to active drug substances, for example, aspirin, warfarin, phenylbutazone, ibuprofen, suprofen, fenbufen, ketoprofen, (S) - (+) - pranoprofen, carprofen, dansylsarcosine, 2 , 3 , 5-triiodobenzoic acid, flufenamic acid, folinic acid, a benzothiadiazide, chlorothiazide, a diazepine, indomethicin, a barbiturate, a cephalosporin, a sulfa drug, an antidiabetic, an antibacterial or an antibiotic.
  • active drug substances for example, aspirin, warfarin, phenylbutazone, ibuprofen, suprofen, fenbufen, ketoprofen, (S) - (+) - pranoprofen
  • Oligonucleotide-drug conjugates and their preparation are described in United States Patent Application 09/334,130 (filed June 15, 1999) which is incorporated herein by reference in its entirety.
  • Representative United States patents that teach the preparation of such oligonucleotide conjugates include, but are not limited to, U.S.: 4,828,979; 4,948,882; 5,218,105;
  • the present invention also includes antisense compounds which are chimeric compounds.
  • "Chimeric” antisense compounds or “chimeras, " in the context of this invention, are antisense compounds, particularly oligonucleotides, which contain two or more chemically distinct regions, each made up of at least one monomer unit, i.e., a nucleotide in the case of an oligonucleotide compound.
  • oligonucleotides typically contain at least one region wherein the oligonucleotide is modified so as to confer upon the oligonucleotide increased resistance to nuclease degradation, increased cellular uptake, increased stability and/or increased binding affinity for the target nucleic acid.
  • An additional region of the oligonucleotide may serve as a substrate for enzymes capable of cleaving RNA: DNA or RNA:RNA hybrids.
  • RNAse H is a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex.
  • RNA:RNA hybrids can, in like fashion, be accomplished through the actions of endoribonucleases, such as interferon- induced RNAseL which cleaves both cellular and viral RNA.
  • RNA target can be routinely detected by gel electrophoresis and, if necessary, associated nucleic acid hybridization techniques known in the art .
  • Chimeric antisense compounds of the invention may be formed as composite structures of two or more oligonucleotides, modified oligonucleotides, oligonucleosides and/or oligonucleotide mimetics as described above. Such compounds have also been referred to in the art as hybrids or gapmers.
  • the compounds of the invention may also be admixed, encapsulated, conjugated or otherwise associated with other molecules, molecule structures or mixtures of compounds, as for example, liposomes, receptor-targeted molecules, oral, rectal, topical or other formulations, for assisting in uptake, distribution and/or absorption.
  • the antisense compounds of the invention encompass any pharmaceutically acceptable salts, esters, or salts of such esters, or any other compound which, upon administration to an animal, including a human, is capable of providing (directly or indirectly) the biologically active metabolite or residue thereof. Accordingly, for example, the disclosure is also drawn to prodrugs and pharmaceutically acceptable salts of the compounds of the invention, pharmaceutically acceptable salts of such prodrugs, and other bioequivalents .
  • prodrug indicates a therapeutic agent that is prepared in an inactive form that is converted to an active form (i.e., drug) within the body or cells thereof by the action of endogenous enzymes or other chemicals and/or conditions.
  • prodrug versions of the oligonucleotides of the invention are prepared as SATE [ (S-acetyl-2-thioethyl) phosphate] derivatives according to the methods disclosed in WO 93/24510 to Gosselin et al . , published December 9, 1993 or in WO 94/26764 and U.S. 5,770,713 to Imbach et al .
  • pharmaceutically acceptable salts refers to physiologically and pharmaceutically acceptable salts of the compounds of the invention: i.e., salts that retain the desired biological activity of the parent compound and do not impart undesired toxicological effects thereto.
  • Pharmaceutically acceptable base addition salts are formed with metals or amines, such as alkali and alkaline earth metals or organic amines.
  • metals or amines such as alkali and alkaline earth metals or organic amines.
  • metals used as cations are sodium, potassium, magnesium, calcium, and the like.
  • suitable amines are sodium, potassium, magnesium, calcium, and the like.
  • the base addition salts of said acidic compounds are prepared by contacting the free acid form with a sufficient amount of the desired base to produce the salt in the conventional manner.
  • the free acid form may be regenerated by contacting the salt form with an acid and isolating the free acid in the conventional manner.
  • the free acid forms differ from their respective salt forms somewhat in certain physical properties such as solubility in polar solvents, but otherwise the salts are equivalent to their respective free acid for purposes of the present invention.
  • pharmaceutical addition salt includes a pharmaceutically acceptable salt of an acid form of one of the components of the compositions of the invention. These include organic or inorganic acid salts of the amines. Preferred acid salts are the hydrochlorides, acetates, salicylates, nitrates and phosphates.
  • Suitable pharmaceutically acceptable salts include basic salts of a variety of inorganic and organic acids, such as, for example, with inorganic acids, such as for example hydrochloric acid, hydrobromic acid, sulfuric acid or phosphoric acid; with organic carboxylic, sulfonic, sulfo or phospho acids or N-substituted sulfamic acids, for example acetic acid, propionic acid, glycolic acid, succinic acid, maleic acid, hydroxymaleic acid, methylmaleic acid, fumaric acid, malic acid, tartaric acid, lactic acid, oxalic acid, gluconic acid, glucaric acid, glucuronic acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, salicylic acid, 4-aminosalicylic acid, 2-phenoxybenzoic acid, 2-acetoxybenzoic acid, embonic acid, nicotinic acid or isonicotin
  • 2-hydroxyethanesulfonic acid ethane-1, 2-disulfonic acid, benzenesulfonic acid, 4-methylbenzenesulfonic acid, napht alene-2-sulfonic acid, naphthalene-1, 5-disulfonic acid, 2- or 3-phosphoglycerate, glucose-6-phosphate, N-cyclohexylsulfamic acid (with the formation of cyclamates) , or with other acid organic compounds, such as ascorbic acid.
  • Pharmaceutically acceptable salts of compounds may also be prepared with a pharmaceutically acceptable cation. Suitable pharmaceutically acceptable cations are well known to those skilled in the art and include alkaline, alkaline earth, ammonium and quaternary ammonium cations. Carbonates or hydrogen carbonates are also possible.
  • salts formed with cations such as sodium, potassium, ammonium, magnesium, calcium, polyamines such as spermine and spermidine, etc.
  • acid addition salts formed with inorganic acids for example hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid and the like
  • salts formed with organic acids such as, for example, acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid, palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonic acid, polygalacturonic acid
  • the antisense compounds of the present invention can be utilized for diagnostics, therapeutics, prophylaxis and as research reagents and kits.
  • an animal preferably a human, suspected of having a disease or disorder which can be treated by modulating the expression of G protein-coupled receptor kinase 6 is treated by administering antisense compounds in accordance with this invention.
  • the compounds of the invention can be utilized in pharmaceutical compositions by adding an effective amount of an antisense compound to a suitable pharmaceutically acceptable diluent or carrier.
  • Use of the antisense compounds and methods of the invention may also be useful prophylactically, e.g., to prevent or delay infection, inflammation or tumor formation, for example .
  • the antisense compounds of the invention are useful for research and diagnostics, because these compounds hybridize to nucleic acids encoding G protein-coupled receptor kinase 6, enabling sandwich and other assays to easily be constructed to exploit this fact.
  • Hybridization of the antisense oligonucleotides of the invention with a nucleic acid encoding G protein-coupled receptor kinase 6 can be detected by means known in the art. Such means may include conjugation of an enzyme to the oligonucleotide, radiolabelling of the oligonucleotide or any other suitable detection means. Kits using such detection means for detecting the level of G protein-coupled receptor kinase 6 in a sample may also be prepared.
  • the present invention also includes pharmaceutical compositions and formulations which include the antisense compounds of the invention.
  • the pharmaceutical compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical (including ophthalmic and to mucous membranes including vaginal and rectal delivery), pulmonary, e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal) , oral or parenteral.
  • Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial , e.g., intrathecal or intraventricular, administration.
  • Oligonucleotides with at least one 2 ' -O- methoxyethyl modification are believed to be particularly useful for oral administration.
  • compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders.
  • Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.
  • Coated condoms, gloves and the like may also be useful .
  • Preferred topical formulations include those in which the oligonucleotides of the invention are in admixture with a topical delivery agent such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents and surfactants.
  • Preferred lipids and liposomes include neutral (e.g.
  • dioleoylphosphatidyl DOPE ethanolamine dimyristoylphosphatidyl choline DMPC, distearolyphosphatidyl choline) negative (e.g. dimyristoylphosphatidyl glycerol DMPG) and cationic (e.g. dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidyl ethanolamine DOTMA) .
  • Oligonucleotides of the invention may be encapsulated within liposomes or may form complexes thereto, in particular to cationic liposomes.
  • oligonucleotides may be complexed to lipids, in particular to cationic lipids.
  • Preferred fatty acids and esters include but are not limited arachidonic acid, oleic acid, eicosanoic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, l-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or a C ] __ ] _ Q alkyl ester (e.g. isopropylmyristate
  • compositions and formulations for oral administration include powders or granules, microparticulates, nanoparticulates, suspensions or solutions in water or non- aqueous media, capsules, gel capsules, sachets, tablets or minitablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable.
  • Preferred oral formulations are those in which oligonucleotides of the invention are administered in conjunction with one or more penetration enhancers surfactants and chelators .
  • Preferred surfactants include fatty acids and/or esters or salts thereof, bile acids and/or salts thereof.
  • Preferred bile acids/salts include chenodeoxycholic acid (CDCA) and ursodeoxychenodeoxycholic acid (UDCA) , cholic acid, dehydrocholic acid, deoxycholic acid, glucholic acid, glycholic acid, glycodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, sodium tauro-24 , 25-dihydro-fusidate and sodium glycodihydrofusidate.
  • DCA chenodeoxycholic acid
  • UDCA ursodeoxychenodeoxycholic acid
  • Preferred fatty acids include arachidonic acid, undecanoic acid, oleic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1- dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or a monoglyceride, a diglyceride or a pharmaceutically acceptable salt thereof (e.g. sodium) .
  • arachidonic acid arachidonic acid, undecanoic acid, oleic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, gly
  • penetration enhancers for example, fatty acids/salts in combination with bile acids/salts.
  • a particularly preferred combination is the sodium salt of lauric acid, capric acid and UDCA.
  • Further penetration enhancers include polyoxyethylene- 9-lauryl ether, polyoxyethylene-20-cetyl ether. Oligonucleotides of the invention may be delivered orally, in granular form including sprayed dried particles, or complexed to form micro or nanoparticles.
  • Oligonucleotide complexing agents include poly-amino acids; polyimines; polyacrylates; polyalkylacrylates, polyoxethanes, polyalkylcyanoacrylates; cationized gelatins, albumins, starches, acrylates, polyethyleneglycols (PEG) and starches; polyalkylcyanoacrylates; DEAE-derivatized polyimines, pollulans, celluloses and starches.
  • Particularly preferred complexing agents include chitosan, N-trimethylchitosan, poly- L-lysine, polyhistidine, polyornithine, polyspermines, protamine, polyvinylpyridine, polythiodiethylamino- methylethylene P(TDAE), polyaminostyrene (e.g.
  • polystyrene resin poly(methylcyanoacrylate) , poly (ethylcyanoacrylate) , poly (butylcyanoacrylate) , poly (isobutylcyanoacrylate) , poly (isohexylcynaoacrylate) , DEAE-methacrylate, DEAE- hexylacrylate, DEAE-acrylamide, DEAE-albumin and DEAE-dextran, polymethylacrylate, polyhexylacrylate, poly (D, L-lactic acid), poly (DL-lactic-co-glycolic acid (PLGA) , alginate, and polyethyleneglycol (PEG) .
  • D L-lactic acid
  • PLGA poly (DL-lactic-co-glycolic acid
  • PEG polyethyleneglycol
  • compositions and formulations for parenteral, intrathecal or intraventricular administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives such as, but not limited to, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients .
  • compositions of the present invention include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions may be generated from a variety of components that include, but are not limited to, preformed liquids, self-emulsifying solids and self-emulsifying semisolids.
  • compositions of the present invention may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier (s) or excipient (s) . In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.
  • the compositions of the present invention may be formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, gel capsules, liquid syrups, soft gels, suppositories, and enemas.
  • the compositions of the present invention may also be formulated as suspensions in aqueous, non-aqueous or mixed media.
  • Aqueous suspensions may further contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran.
  • the suspension may also contain stabilizers.
  • the pharmaceutical compositions may be formulated and used as foams.
  • Pharmaceutical foams include formulations such as, but not limited to, emulsions, microemulsions, creams, jellies and liposomes. While basically similar in nature these formulations vary in the components and the consistency of the final product. The preparation of such compositions and formulations is generally known to those skilled in the pharmaceutical and formulation arts and may be applied to the formulation of the compositions of the present invention.
  • compositions of the present invention may be prepared and formulated as emulsions.
  • Emulsions are typically heterogenous systems of one liquid dispersed in another in the form of droplets usually exceeding 0.1 ⁇ m in diameter (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y. , Volume 1, p.
  • Emulsions are often biphasic systems comprising two immiscible liquid phases intimately mixed and dispersed with each other.
  • emulsions may be of either the water-in-oil (w/o) or the oil-in-water (o/w) variety.
  • Emulsions may contain additional components in addition to the dispersed phases, and the active drug which may be present as a solution in either the aqueous phase, oily phase or itself as a separate phase. Pharmaceutical excipients such as emulsifiers, stabilizers, dyes, and antioxidants may also be present in emulsions as needed.
  • compositions may also be multiple emulsions that are comprised of more than two phases such as, for example, in the case of oil-in-water-in-oil (o/w/o) and water-in-oil-in- water (w/o/w) emulsions.
  • Such complex formulations often provide certain advantages that simple binary emulsions do not.
  • Multiple emulsions in which individual oil droplets of an o/w emulsion enclose small water droplets constitute a w/o/w emulsion.
  • a system of oil droplets enclosed in globules of water stabilized in an oily continuous phase provides an o/w/o emulsion.
  • Emulsions are characterized by little or no thermodynamic stability. Often, the dispersed or discontinuous phase of the emulsion is well dispersed into the external or continuous phase and maintained in this form through the means of emulsifiers or the viscosity of the formulation. Either of the phases of the emulsion may be a semisolid or a solid, as is the case of emulsion-style ointment bases and creams. Other means of stabilizing emulsions entail the use of emulsifiers that may be incorporated into either phase of the emulsion. Emulsifiers may broadly be classified into four categories: synthetic surfactants, naturally occurring emulsifiers, absorption bases, and finely dispersed solids
  • Synthetic surfactants also known as surface active agents, have found wide applicability in the formulation of emulsions and have been reviewed in the literature (Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y. , volume 1, p. 285; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), Marcel Dekker, Inc., New York, N.Y., 1988, volume 1, p. 199) .
  • Surfactants are typically amphiphilic and comprise a hydrophilic and a hydrophobic portion.
  • HLB hydrophile/lipophile balance
  • surfactants may be classified into different classes based on the nature of the hydrophilic group: nonionic, anionic, cationic and amphoteric (Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker
  • Naturally occurring emulsifiers used in emulsion formulations include lanolin, beeswax, phosphatides, lecithin and acacia.
  • polar inorganic solids such as heavy metal hydroxides, nonswelling clays such as bentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidal aluminum silicate and colloidal magnesium aluminum silicate, pigments and nonpolar solids such as carbon or glyceryl tristearate.
  • non-emulsifying materials are also included in emulsion formulations and contribute to the properties of emulsions.
  • Hydrophilic colloids or hydrocolloids include naturally occurring gums and synthetic polymers such as polysaccharides
  • cellulose derivatives for example, carboxymethylcellulose and carboxypropylcellulose
  • synthetic polymers for example, carbomers, cellulose ethers, and carboxyvinyl polymers
  • emulsions often contain a number of ingredients such as carbohydrates, proteins, sterols and phosphatides that may readily support the growth of microbes, these formulations often incorporate preservatives.
  • preservatives included in emulsion formulations include methyl paraben, propyl paraben, quaternary ammonium salts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boric acid.
  • Antioxidants are also commonly added to emulsion formulations to prevent deterioration of the formulation.
  • Antioxidants used may be free radical scavengers such as tocopherols, alkyl gallates, butylated hydroxyanisole, butylated hydroxytoluene , or reducing agents such as ascorbic acid and sodium metabisulfite, and antioxidant synergists such as citric acid, tartaric acid, and lecithin.
  • free radical scavengers such as tocopherols, alkyl gallates, butylated hydroxyanisole, butylated hydroxytoluene , or reducing agents such as ascorbic acid and sodium metabisulfite
  • antioxidant synergists such as citric acid, tartaric acid, and lecithin.
  • Emulsion formulations for oral delivery have been very widely used because of ease of formulation, as well as efficacy from an absorption and bioavailability standpoint (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p.
  • compositions of oligonucleotides and nucleic acids are formulated as microemulsions.
  • a microemulsion may be defined as a system of water, oil and amphiphile which is a single optically isotropic and thermodynamically stable liquid solution (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245) .
  • Typical microemulsions are systems that are prepared by first dispersing an oil in an aqueous surfactant solution and then adding a sufficient amount of a fourth component, generally an intermediate chain- length alcohol to form a transparent system.
  • microemulsions have also been described as thermodynamically stable, isotropically clear dispersions of two immiscible liquids that are stabilized by interfacial films of surface- active molecules (Leung and Shah, in : Controlled Release of Drugs : Polymers and Aggregate Systems, Rosoff, M. , Ed., 1989, VCH Publishers, New York, pages 185-215) .
  • Microemulsions commonly are prepared via a combination of three to five components that include oil, water, surfactant, cosurfactant and electrolyte.
  • microemulsion is of the water- in-oil (w/o) or an oil-in-water (o/w) type is dependent on the properties of the oil and surfactant used and on the structure and geometric packing of the polar heads and hydrocarbon tails of the surfactant molecules (Schott, in Remington ' s
  • microemulsions offer the advantage of solubilizing water-insoluble drugs in a formulation of thermodynamically stable droplets that are formed spontaneously.
  • Surfactants used in the preparation of microemulsions include, but are not limited to, ionic surfactants, non-ionic surfactants, Brij 96, polyoxyethylene oleyl ethers, polyglycerol fatty acid esters, tetraglycerol monolaurate (ML310) , tetraglycerol monooleate (MO310) , hexaglycerol monooleate (PO310) , hexaglycerol pentaoleate (PO500) , decaglycerol monocaprate (MCA750) , decaglycerol monooleate (MO750), decaglycerol sequioleate (SO750) , decaglycerol decaoleate (DAO750) , alone or in combination with cosurfactants .
  • ionic surfactants non-ionic surfactants
  • Brij 96 polyoxyethylene oleyl ethers
  • polyglycerol fatty acid esters
  • the cosurfactant usually a short-chain alcohol such as ethanol, 1-propanol, and 1-butanol, serves to increase the interfacial fluidity by penetrating into the surfactant film and consequently creating a disordered film because of the void space generated among surfactant molecules.
  • Microemulsions may, however, be prepared without the use of cosurfactants and alcohol-free self-emulsifying microemulsion systems are known in the art.
  • the aqueous phase may typically be, but is not limited to, water, an aqueous solution of the drug, glycerol, PEG300, PEG400, polyglycerols, propylene glycols, and derivatives of ethylene glycol.
  • the oil phase may include, but is not limited to, materials such as Captex 300, Captex 355, Capmul MCM, fatty acid esters, medium chain (C8-C12) mono, di, and tri-glycerides, polyoxyethylated glyceryl fatty acid esters, fatty alcohols, polyglycolized glycerides, saturated polyglycolized C8-C10 glycerides, vegetable oils and silicone oil.
  • Microemulsions are particularly of interest from the standpoint of drug solubilization and the enhanced absorption of drugs. Lipid based microemulsions (both o/w and w/o) have been proposed to enhance the oral bioavailability of drugs, including peptides (Constantinides et al .
  • Microemulsions afford advantages of improved drug solubilization, protection of drug from enzymatic hydrolysis, possible enhancement of drug absorption due to surfactant-induced alterations in membrane fluidity and permeability, ease of preparation, ease of oral administration over solid dosage forms, improved clinical potency, and decreased toxicity (Constantinides et al . , Pharmaceutical Research, 1994, 11 , 1385; Ho et al . , J. Pharm . Sci . , 1996, 85, 138-143).
  • microemulsions may form spontaneously when their components are brought together at ambient temperature. This may be particularly advantageous when formulating thermolabile drugs, peptides or oligonucleotides.
  • Microemulsions have also been effective in the transdermal delivery of active components in both cosmetic and pharmaceutical applications. It is expected that the microemulsion compositions and formulations of the present invention will facilitate the increased systemic absorption of oligonucleotides and nucleic acids from the gastrointestinal tract, as well as improve the local cellular uptake of oligonucleotides and nucleic acids within the gastrointestinal tract, vagina, buccal cavity and other areas of administration.
  • Microemulsions of the present invention may also contain additional components and additives such as sorbitan monostearate (Grill 3), Labrasol, and penetration enhancers to improve the properties of the formulation and to enhance the absorption of the oligonucleotides and nucleic acids of the present invention.
  • Penetration enhancers used in the microemulsions of the present invention may be classified as belonging to one of five broad categories - surfactants, fatty acids, bile salts, chelating agents, and non-chelating non- surfactants (Lee et al . , Cri tical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92) . Each of these classes has been discussed above.
  • Liposomes There are many organized surfactant structures besides microemulsions that have been studied and used for the formulation of drugs. These include monolayers, micelles, bilayers and vesicles. Vesicles, such as liposomes, have attracted great interest because of their specificity and the duration of action they offer from the standpoint of drug delivery.
  • liposome means a vesicle composed of amphiphilic lipids arranged in a spherical bilayer or bilayers.
  • Liposomes are unilamellar or multilamellar vesicles which have a membrane formed from a lipophilic material and an aqueous interior. The aqueous portion contains the composition to be delivered. Cationic liposomes possess the advantage of being able to fuse to the cell wall. Non- cationic liposomes, although not able to fuse as efficiently with the cell wall, are taken up by macrophages in vivo .
  • lipid vesicles In order to cross intact mammalian skin, lipid vesicles must pass through a series of fine pores, each with a diameter less than 50 nm, under the influence of a suitable transdermal gradient. Therefore, it is desirable to use a liposome which is highly deformable and able to pass through such fine pores.
  • liposomes obtained from natural phospholipids are biocompatible and biodegradable; liposomes can incorporate a wide range of water and lipid soluble drugs; liposomes can protect encapsulated drugs in their internal compartments from metabolism and degradation (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y. , volume 1, p. 245) .
  • Important considerations in the preparation of liposome formulations are the lipid surface charge, vesicle size and the aqueous volume of the liposomes.
  • Liposomes are useful for the transfer and delivery of active ingredients to the site of action. Because the liposomal membrane is structurally similar to biological membranes, when liposomes are applied to a tissue, the liposomes start to merge with the cellular membranes and as the merging of the liposome and cell progresses, the liposomal contents are emptied into the cell where the active agent may act .
  • Liposomes present several advantages over other formulations. Such advantages include reduced side-effects related to high systemic absorption of the administered drug, increased accumulation of the administered drug at the desired target, and the ability to administer a wide variety of drugs, both hydrophilic and hydrophobic, into the skin.
  • liposomes to deliver agents including high-molecular weight DNA into the skin.
  • Compounds including analgesics, antibodies, hormones and high-molecular weight DNAs have been administered to the skin. The majority of applications resulted in the targeting of the upper epidermis.
  • Liposomes fall into two broad classes. Cationic liposomes are positively charged liposomes which interact with the negatively charged DNA molecules to form a stable complex. The positively charged DNA/liposome complex binds to the negatively charged cell surface and is internalized in an endosome . Due to the acidic pH within the endosome, the liposomes are ruptured, releasing their contents into the cell cytoplasm (Wang et al . , Biochem . Biophys . Res . Commun . , 1987, 147, 980-985) .
  • Liposomes which are pH-sensitive or negatively-charged, entrap DNA rather than complex with it. Since both the DNA and the lipid are similarly charged, repulsion rather than complex formation occurs. Nevertheless, some DNA is entrapped within the aqueous interior of these liposomes. pH-sensitive liposomes have been used to deliver DNA encoding the thymidine kinase gene to cell monolayers in culture. Expression of the exogenous gene was detected in the target cells (Zhou et al . , Journal of Controlled Release, 1992, 19, 269-274) .
  • liposomal composition includes phospholipids other than naturally-derived phosphatidylcholine .
  • Neutral liposome compositions for example, can be formed from dimyristoyl phosphatidylcholine
  • DMPC dimyristoyl phosphatidylglycerol
  • anionic fusogenic liposomes are formed primarily from dioleoyl phosphatidylethanolamine (DOPE) .
  • DOPE dioleoyl phosphatidylethanolamine
  • Another type of liposomal composition is formed from phosphatidylcholine (PC) such as, for example, soybean PC, and egg PC.
  • PC phosphatidylcholine
  • Another type is formed from mixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.
  • Non-ionic liposomal systems have also been examined to determine their utility in the delivery of drugs to the skin, in particular systems comprising non-ionic surfactant and cholesterol.
  • Non-ionic liposomal formulations comprising NovasomeTM I (glyceryl dilaurate/cholesterol/polyoxyethylene- 10-stearyl ether) and NovasomeTM II (glyceryl distearate/ cholesterol/polyoxyethylene-10-stearyl ether) were used to deliver cyclosporin-A into the dermis of mouse skin. Results indicated that such non-ionic liposomal systems were effective in facilitating the deposition of cyclosporin-A into different layers of the skin (Hu et al . S . T. P. Pharma . Sci .
  • Liposomes also include "sterically stabilized" liposomes, a term which, as used herein, refers to liposomes comprising one or more specialized lipids that, when incorporated into liposomes, result in enhanced circulation lifetimes relative to liposomes lacking such specialized lipids.
  • sterically stabilized liposomes are those in which part of the vesicle-forming lipid portion of the liposome (A) comprises one or more glycolipids, such as monosialoganglioside G M ⁇ , or (B) is derivatized with one or more hydrophilic polymers, such as a polyethylene glycol (PEG) moiety.
  • PEG polyethylene glycol
  • liposomes comprising (1) sphingomyelin and (2) the ganglioside G MI or a galactocerebroside sulfate ester.
  • U.S. Patent No. 5,543,152 discloses liposomes comprising sphingomyelin. Liposomes comprising 1 , 2 - sn- dimyristoylphosphatidylcholine are disclosed in WO 97/13499 (Lim et al . ) .
  • liposomes comprising lipids derivatized with one or more hydrophilic polymers, and methods of preparation thereof, are known in the art.
  • Sunamoto et al . ⁇ Bull . Chem. Soc . Jpn . , 1980, 53 , 2778 described liposomes comprising a nonionic detergent, 2C 12 15G, that contains a PEG moiety.
  • Ilium et al . FEBS Lett . , 1984, 167, 79
  • hydrophilic coating of polystyrene particles with polymeric glycols results in significantly enhanced blood half-lives.
  • DSPE-PEG formed from the combination of distearoylphosphatidylethanolamine (DSPE) and PEG.
  • DSPE distearoylphosphatidylethanolamine
  • PEG distearoylphosphatidylethanolamine
  • Liposomes having covalently bound PEG moieties on their external surface are described in European Patent No. EP 0 445 131 Bl and WO 90/04384 to Fisher.
  • Liposome compositions containing 1-20 mole percent of PE derivatized with PEG, and methods of use thereof, are described by Woodle et al . (U.S. Patent Nos. 5,013,556 and 5,356,633) and Martin et al . (U.S. Patent No.
  • WO 96/40062 to Thierry et al discloses methods for encapsulating high molecular weight nucleic acids in liposomes.
  • U.S. Patent No. 5,264,221 to Tagawa et al discloses protein-bonded liposomes and asserts that the contents of such liposomes may include an antisense RNA.
  • U.S. Patent No. 5,665,710 to Rahman et al describes certain methods of encapsulating oligodeoxynucleotides in liposomes.
  • WO 97/04787 to Love et al discloses liposomes comprising antisense oligonucleotides targeted to the raf gene.
  • Transfersomes are yet another type of liposomes, and are highly deformable lipid aggregates which are attractive candidates for drug delivery vehicles. Transfersomes may be described as lipid droplets which are so highly deformable that they are easily able to penetrate through pores which are smaller than the droplet. Transfersomes are adaptable to the environment in which they are used, e . g. they are self- optimizing (adaptive to the shape of pores in the skin) , self- repairing, frequently reach their targets without fragmenting, and often self-loading.
  • Transfersomes have been used to deliver serum albumin to the skin.
  • the transfersome- mediated delivery of serum albumin has been shown to be as effective as subcutaneous injection of a solution containing serum albumin.
  • HLB hydrophile/lipophile balance
  • Nonionic surfactants find wide application in pharmaceutical and cosmetic products and are usable over a wide range of pH values. In general their HLB values range from 2 to about 18 depending on their structure.
  • Nonionic surfactants include nonionic esters such as ethylene glycol esters, propylene glycol esters, glyceryl esters, polyglyceryl esters, sorbitan esters, sucrose esters, and ethoxylated esters.
  • Nonionic alkanolamides and ethers such as fatty alcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylated block polymers are also included in this class.
  • the polyoxyethylene surfactants are the most popular members of the nonionic surfactant class.
  • Anionic surfactants include carboxylates such as soaps, acyl lactylates, acyl amides of amino acids, esters of sulfuric acid such as alkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkyl benzene sulfonates, acyl isethionates, acyl taurates and sulfosuccinates, and phosphates.
  • the most important members of the anionic surfactant class are the alkyl sulfates and the soaps .
  • Cationic surfactants include quaternary ammonium salts and ethoxylated amines. The quaternary ammonium salts are the most used members of this class .
  • amphoteric surfactants include acrylic acid derivatives, substituted alkylamides, N- alkylbetaines and phosphatides.
  • the present invention employs various penetration enhancers to effect the efficient delivery of nucleic acids, particularly oligonucleotides, to the skin of animals.
  • nucleic acids particularly oligonucleotides
  • Most drugs are present in solution in both ionized and nonionized forms. However, usually only lipid soluble or lipophilic drugs readily cross cell membranes. It has been discovered that even non-lipophilic drugs may cross cell membranes if the membrane to be crossed is treated with a penetration enhancer. In addition to aiding the diffusion of non-lipophilic drugs across cell membranes, penetration enhancers also enhance the permeability of lipophilic drugs.
  • Penetration enhancers may be classified as belonging to one of five broad categories, i.e., surfactants, fatty acids, bile salts, chelating agents, and non-chelating non- surfactants (Lee et al . , Cri tical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92) . Each of the above mentioned classes of penetration enhancers are described below in greater detail .
  • surfactants are chemical entities which, when dissolved in an aqueous solution, reduce the surface tension of the solution or the interfacial tension between the aqueous solution and another liquid, with the result that absorption of oligonucleotides through the mucosa is enhanced.
  • these penetration enhancers include, for example, sodium lauryl sulfate, polyoxyethylene-9-lauryl ether and polyoxyethylene- 20-cetyl ether) (Lee et al . , Cri tical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92); and perfluorochemical emulsions, such as FC-43. Takahashi et al . , J " . Pharm . Pharmacol . , 1988, 40, 252).
  • Fatty acids Various fatty acids and their derivatives which act as penetration enhancers include, for example, oleic acid, lauric acid, capric acid (n-decanoic acid) , myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein (1-monooleoyl-rac- glycerol) , dilaurin, caprylic acid, arachidonic acid, glycerol 1-monocaprate, l-dodecylazacycloheptan-2-one, acylcarnitines, acylcholines, d- 10 alkyl esters thereof ⁇ e .
  • oleic acid lauric acid
  • capric acid n-decanoic acid
  • myristic acid palmitic acid
  • stearic acid linoleic acid
  • linolenic acid dicaprate
  • tricaprate
  • Bile salts The physiological role of bile includes the facilitation of dispersion and absorption of lipids and fat- soluble vitamins (Brunton, Chapter 38 in: Goodman & Gilman's The Pharmacological Basis of Therapeutics, 9th Ed. , Hardman et al . Eds., McGraw-Hill, New York, 1996, pp. 934-935).
  • the term "bile salts" includes any of the naturally occurring components of bile as well as any of their synthetic derivatives.
  • the bile salts of the invention include, for example, cholic acid (or its pharmaceutically acceptable sodium salt, sodium cholate) , dehydrocholic acid (sodium dehydrocholate) , deoxycholic acid (sodium deoxycholate) , glucholic acid (sodium glucholate) , glycholic acid (sodium glycocholate) , glycodeoxycholic acid (sodium glycodeoxycholate) , taurocholic acid (sodium taurocholate) , taurodeoxycholic acid (sodium taurodeoxycholate) , chenodeoxycholic acid (sodium chenodeoxycholate) , ursodeoxycholic acid (UDCA) , sodium tauro- 24, 25-dihydro-fusidate (STDHF), sodium glycodihydrofusidate and polyoxyethylene-9-lauryl ether (POE) (Lee et al .
  • cholic acid or its pharmaceutically
  • Chelating agents can be defined as compounds that remove metallic ions from solution by forming complexes therewith, with the result that absorption of oligonucleotides through the mucosa is enhanced.
  • chelating agents have the added advantage of also serving as DNase inhibitors, as most characterized DNA nucleases require a divalent metal ion for catalysis and are thus inhibited by chelating agents (Jarrett, J " . Chromatogr . , 1993, 618 , 315-339) .
  • Chelating agents of the invention include but are not limited to disodium ethylenediaminetetraacetate (EDTA) , citric acid, salicylates [ e . g. , sodium salicylate, 5-methoxysalicylate and homovanilate) , iV-acyl derivatives of collagen, laureth-9 and iV-amino acyl derivatives of beta-diketones (enamines) (Lee et al . , Cri tical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92; Muranishi, Cri tical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Buur et al . , J. Control Rel . , 1990, 14 , 43-51) .
  • EDTA disodium ethylenediaminetetraacetate
  • citric acid citric acid
  • salicylates e . g. , sodium salicylate, 5-methoxysalicylate and homovanilate
  • Non-chelating non-surfactants can be defined as compounds that demonstrate insignificant activity as chelating agents or as surfactants but that nonetheless enhance absorption of oligonucleotides through the alimentary mucosa (Muranishi, Cri tical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33) .
  • This class of penetration enhancers include, for example, unsaturated cyclic ureas, 1-alkyl- and 1-alkenylazacyclo-alkanone derivatives (Lee et al .
  • non-steroidal anti-inflammatory agents such as diclofenac sodium, indomethacin and phenylbutazone (Yamashita et al . , J. Pharm. Pharmacol . , 1987, 39, 621-626) .
  • Agents that enhance uptake of oligonucleotides at the cellular level may also be added to the pharmaceutical and other compositions of the present invention.
  • cationic lipids such as lipofectin (Junichi et al , U.S. Patent No. 5,705,188), cationic glycerol derivatives, and polycationic molecules, such as polylysine (Lollo et al . , PCT Application WO 97/30731) , are also known to enhance the cellular uptake of oligonucleotides.
  • agents may be utilized to enhance the penetration of the administered nucleic acids, including glycols such as ethylene glycol and propylene glycol, pyrrols such as 2- pyrrol, azones, and terpenes such as limonene and menthone.
  • glycols such as ethylene glycol and propylene glycol
  • pyrrols such as 2- pyrrol
  • azones such as 2- pyrrol
  • terpenes such as limonene and menthone.
  • compositions of the present invention also incorporate carrier compounds in the formulation.
  • carrier compound or “carrier” can refer to a nucleic acid, or analog thereof, which is inert (i.e., does not possess biological activity per se) but is recognized as a nucleic acid by in vivo processes that reduce the bioavailability of a nucleic acid having biological activity by, for example, degrading the biologically active nucleic acid or promoting its removal from circulation.
  • a nucleic acid and a carrier compound can result in a substantial reduction of the amount of nucleic acid recovered in the liver, kidney or other extracirculatory reservoirs, presumably due to competition between the carrier compound and the nucleic acid for a common receptor.
  • the recovery of a partially phosphorothioate oligonucleotide in hepatic tissue can be reduced when it is coadministered with polyinosinic acid, dextran sulfate, polycytidic acid or 4-acetamido-4 ' isothiocyano-stilbene-2 , 2 ' - disulfonic acid (Miyao et al . , Antisense Res . Dev. , 1995, 5, 115-121; Takakura et al . , Antisense & Nucl . Acid Drug Dev. , 1996, 6, 177-183) .
  • a "pharmaceutical carrier” or “excipient” is a pharmaceutically acceptable solvent, suspending agent or any other pharmacologically inert vehicle for delivering one or more nucleic acids to an animal .
  • the excipient may be liquid or solid and is selected, with the planned manner of administration in mind, so as to provide for the desired bulk, consistency, etc., when combined with a nucleic acid and the other components of a given pharmaceutical composition.
  • Typical pharmaceutical carriers include, but are not limited to, binding agents ⁇ e . g. , pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers ( e . g.
  • lubricants e.g., magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, corn starch, polyethylene glycols, sodium benzoate, sodium acetate, etc.
  • disintegrants e . g. , starch, sodium starch glycolate, etc.
  • wetting agents e . g. , sodium lauryl sulphate, etc.
  • compositions of the present invention can also be used to formulate the compositions of the present invention.
  • suitable pharmaceutically acceptable carriers include, but are not limited to, water, salt solutions, alcohols, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like.
  • Formulations for topical administration of nucleic acids may include sterile and non-sterile aqueous solutions, non- aqueous solutions in common solvents such as alcohols, or solutions of the nucleic acids in liquid or solid oil bases.
  • the solutions may also contain buffers, diluents and other suitable additives.
  • Pharmaceutically acceptable organic or inorganic excipients suitable for non-parenteral administration which do not deleteriously react with nucleic acids can be used.
  • Suitable pharmaceutically acceptable excipients include, but are not limited to, water, salt solutions, alcohol, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like.
  • compositions of the present invention may additionally contain other adjunct components conventionally found in pharmaceutical compositions, at their art-established usage levels.
  • the compositions may contain additional, compatible, pharmaceutically-active materials such as, for example, antipruritics, astringents, local anesthetics or anti-inflammatory agents, or may contain additional materials useful in physically formulating various dosage forms of the compositions of the present invention, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers.
  • additional materials useful in physically formulating various dosage forms of the compositions of the present invention such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers.
  • auxiliary agents e . g.
  • Aqueous suspensions may contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran.
  • the suspension may also contain stabilizers.
  • Certain embodiments of the invention provide pharmaceutical compositions containing (a) one or more antisense compounds and (b) one or more other chemotherapeutic agents which function by a non-antisense mechanism.
  • chemotherapeutic agents include but are not limited to daunorubicin, daunomycin, dactinomycin, doxorubicin, epirubicin, idarubicin, esorubicin, bleomycin, mafosfamide, ifosfamide, cytosine arabinoside, bis-chloroethylnitrosurea, busulfan, mitomycin C, actinomycin D, mithramycin, prednisone, hydroxyprogesterone, testosterone, tamoxifen, dacarbazine, procarbazine, hexamethylme1amine, pentamethylmelamine, mitoxantrone, amsacrine, chlorambucil, methylcyclohexylnitrosurea, nitrogen mustards, melphalan, cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-azacytidine, hydroxyurea
  • chemotherapeutic agents may be used individually (e . g. , 5-FU and oligonucleotide), sequentially (e . g. , 5-FU and oligonucleotide for a period of time followed by MTX and oligonucleotide) , or in combination with one or more other such chemotherapeutic agents ( e . g.
  • Anti-inflammatory drugs including but not limited to nonsteroidal anti-inflammatory drugs and corticosteroids, and antiviral drugs, including but not limited to ribivirin, vidarabine, acyclovir and ganciclovir, may also be combined in compositions of the invention. See, generally, The Merck Manual of Diagnosis and Therapy, 15th
  • compositions of the invention may contain one or more antisense compounds, particularly oligonucleotides, targeted to a first nucleic acid and one or more additional antisense compounds targeted to a second nucleic acid target.
  • antisense compounds particularly oligonucleotides
  • additional antisense compounds targeted to a second nucleic acid target Numerous examples of antisense compounds are known in the art. Two or more combined compounds may be used together or sequentially.
  • Dosing is dependent on severity and responsiveness of the disease state to be treated, with the course of treatment lasting from several days to several months, or until a cure is effected or a diminution of the disease state is achieved.
  • Optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the patient. Persons of ordinary skill can easily determine optimum dosages, dosing methodologies and repetition rates. Optimum dosages may vary depending on the relative potency of individual oligonucleotides, and can generally be estimated based on EC 50 s found to be effective in in vi tro and in vivo animal models.
  • dosage is from 0.01 ug to 100 g per kg of body weight, and may be given once or more daily, weekly, monthly or yearly, or even once every 2 to 20 years. Persons of ordinary skill in the art can easily estimate repetition rates for dosing based on measured residence times and concentrations of the drug in bodily fluids or tissues.
  • 2 ' -Deoxy and 2 ' -methoxy beta-cyanoethyldiisopropyl phosphoramidites were purchased from commercial sources (e.g. Chemgenes, Needham MA or Glen Research, Inc. Sterling VA) .
  • Other 2 ' -O-alkoxy substituted nucleoside amidites are prepared as described in U.S. Patent 5,506,351, herein incorporated by reference.
  • optimized synthesis cycles were developed that incorporate multiple steps coupling longer wait times relative to standard synthesis cycles.
  • TLC thin layer chromatography
  • MP melting point
  • HPLC high pressure liquid chromatography
  • NMR Nuclear Magnetic Resonance
  • argon Ar
  • MeOH methanol
  • MeOH dichloromethane
  • TAA triethylamine
  • DMF dimethyl formamide
  • EtOAc dimethyl sulfoxide
  • THF tetrahydrofuran
  • Oligonucleotides containing 5-methyl-2 ' -deoxycytidine (5- Me-dC) nucleotides were synthesized according to published methods (Sanghvi, et . al . , Nucleic Acids Research, 1993, 21 , 3197-3203) using commercially available phosphoramidites (Glen Research, Sterling VA or ChemGenes, Needham MA) or prepared as follows :
  • thymidine (1.00 kg, 4.13 mol) in anhydrous pyridine (6 L) at ambient temperature.
  • Dimethoxytrityl (DMT) chloride (1.47 kg, 4.34 mol, 1.05 eq) was added as a solid in four portions over 1 h. After 30 min, TLC indicated approx. 95% product, 2% thymidine, 5% DMT reagent and by-products and 2 % 3', 5' -bis DMT product (R f in EtOAc 0.45, 0.05, 0.98, 0.95 respectively) .
  • Trimethylsilylchloride (2.1 L, 16.5 mol, 3.0 eq) was added over 30 minutes while maintaining the internal temperature below -5°C, followed by a wash of anhydrous acetonitrile (1 L) . Note: the reaction is mildly exothermic and copious hydrochloric acid fumes form over the course of the addition.
  • the reaction. was allowed to warm to 0°C and the reaction progress was confirmed by TLC (EtOAc-hexanes 4:1; R f 0.43 to 0.84 of starting material and silyl product, respectively) .
  • triazole (3.05 kg, 44 mol, 8.0 eq) was added the reaction was cooled to -20 °C internal temperature (external -30°C) .
  • Phosphorous oxychloride (1035 L, 11.1 mol, 2.01 eq) was added over 60 min so as to maintain the temperature between -20°C and -10°C during the strongly exothermic process, followed by a wash of anhydrous acetonitrile (1 L) .
  • the reaction was warmed to 0 °C and stirred for 1 h.
  • TLC indicated a complete conversion to the triazole product (R f 0.83 to 0.34 with the product spot glowing in long wavelength UV light) .
  • the reaction mixture was a peach-colored thick suspension, which turned darker red upon warming without apparent decomposition.
  • reaction was cooled to -15 °C internal temperature and water (5 L) was slowly added at a rate to maintain the temperature below +10 °C in order to quench the reaction and to form a homogenous solution. (Caution: this reaction is initially very strongly exothermic) .
  • Approximately one-half of the reaction volume (22 L) was transferred by air pump to another vessel, diluted with EtOAc (12 L) and extracted with water (2 x 8 L) .
  • the combined water layers were back-extracted with EtOAc (6 L) .
  • the water layer was discarded and the organic layers were concentrated in a 20 L rotary evaporator to an oily foam.
  • the foam was coevaporated with anhydrous acetonitrile (4 L) to remove EtOAc .
  • dioxane may be used instead of anhydrous acetonitrile if dried to a hard foam
  • the second half of the reaction was treated in the same way. Each residue was dissolved in dioxane (3 L) and concentrated ammonium hydroxide (750 mL) was added. A homogenous solution formed in a few minutes and the reaction was allowed to stand overnight (although the reaction is complete within 1 h) .
  • the residue was dissolved in EtOAc (1 L) and yielded a third crop which was treated as above except that more washing was required to remove a yellow oily layer.
  • the three crops were dried in a vacuum oven (50°C, 0.1 mm Hg, 24 h) to a constant weight (1750, 600 and 200 g, respectively) and combined to afford 2550 g (85%) of a white crystalline product (MP 215-217 °C) when TLC and NMR spectroscopy indicated purity.
  • the mother liquor still contained mostly product (as determined by TLC) and a small amount of triazole (as determined by NMR spectroscopy) , bis DMT product and unidentified minor impurities.
  • the mother liquor can be purified by silica gel chromatography using a gradient of MeOH (0-25%) in EtOAc to further increase the yield.
  • Crystalline 5 ' -0-dimethoxytrityl-5-methyl-2 ' - deoxycytidine (2000 g, 3.68 mol) was dissolved in anhydrous DMF (6.0 kg) at ambient temperature in a 50 L glass reactor vessel equipped with an air stirrer and argon line.
  • Benzoic anhydride (Chem Impex not Aldrich, 874 g, 3.86 mol, 1.05 eq) was added and the reaction was stirred at ambient temperature for 8 h.
  • TLC CH 2 C1 2 -EtOAc; CH 2 Cl 2 -EtOAc 4:1; R f 0.25) indicated approx. 92% complete reaction.
  • An additional amount of benzoic anhydride (44 g, 0.19 mol) was added.
  • THe product was purified by Biotage column chromatography (5 kg Biotage) prepared with 65:35:1 hexanes-EtOAc-TEA (4L) .
  • the crude product 800 g
  • dissolved in CH 2 Cl 2 (2 L) was applied to the column.
  • the column was washed with the 65:35:1 solvent mixture (20 kg), then 20:80:1 solvent mixture (10 kg), then 99:1 EtOAc :TEA (17kg).
  • the fractions containing the product were collected, and any fractions containing the product and impurities were retained to be resubjected to column chromatography.
  • the column was re-equilibrated with the original 65:35:1 solvent mixture (17 kg) .
  • a second batch of crude product (840 g) was applied to the column as before.
  • the column was washed with the following solvent gradients: 65:35:1 (9 kg), 55:45:1 (20 kg), 20:80:1 (10 kg), and 99:1 EtOAc : TEA (15 kg).
  • the column was reequilibrated as above, and a third batch of the crude product (850 g) plus impure fractions recycled from the two previous columns (28 g) was purified following the procedure for the second batch.
  • the protected nucleoside N6-benzoyl-2 ' - deoxy-2 ' -fluoroadenosine was synthesized utilizing commercially available 9-beta-D-arabinofuranosyladenine as starting material and whereby the 2 ' -alpha-fluoro atom is introduced by a S N 2 -displacement of a 2 ' -beta-triflate group.
  • N6-benzoyl-9-beta-D-arabinofuranosyladenine was selectively protected in moderate yield as the 3 ' , 5 ' - ditetrahydropyranyl (THP) intermediate.
  • THP 5 ' -dimethoxytrityl-
  • 5'- DMT-3 ' -phosphoramidite intermediates was accomplished using standard methodologies to obtain the 5 ' -dimethoxytrityl- (DMT) and 5'- DMT-3 ' -phosphoramidite intermediates.
  • Synthesis of 2 ' -deoxy-2 ' -fluorouridine was accomplished by the modification of a literature procedure in which 2,2'- anhydro-1-beta-D-arabinofuranosyluracil was treated with 70% hydrogen fluoride-pyridine . Standard procedures were used to obtain the 5 ' -DMT and 5 ' -DMT-3 'phosphoramidites .
  • 2 ' -deoxy-2 ' -fluorocytidine was synthesized via amination of 2 ' -deoxy-2 ' -fluorouridine, followed by selective protection to give N4-benzoyl-2 ' -deoxy-2 ' -fluorocytidine . Standard procedures were used to obtain the 5 ' -DMT and 5 ' -DMT- 3 ' phosphoramidites .
  • the gum was redissolved in brine (3 L) , and the flask was rinsed with additional brine (3 L) .
  • the combined aqueous solutions were extracted with chloroform (20 L) in a heavier- than continuous extractor for 70 h.
  • the chloroform layer was concentrated by rotary evaporation in a 20 L flask to a sticky foam (2400 g) . This was coevaporated with MeOH (400 mL) and EtOAc (8 L) at 75°C and 0.65 at until the foam dissolved at which point the vacuum was lowered to about 0.5 atm. After 2.5 L of distillate was collected a precipitate began to form and the flask was removed from the rotary evaporator and stirred until the suspension reached ambient temperature.
  • EtOAc (2 L) was added and the slurry was filtered on a 25 cm table top Buchner funnel and the product was washed with EtOAc (3 x 2 L) .
  • the bright white solid was air dried in pans for 24 h then further dried in a vacuum oven (50°C, 0.1 mm Hg, 24 h) to afford 1649 g of a white crystalline solid (mp 115.5- 116.5°C) .
  • the brine layer in the 20 L continuous extractor was further extracted for 72 h with recycled chloroform.
  • the chloroform was concentrated to 120 g of oil and this was combined with the mother liquor from the above filtration (225 g) , dissolved in brine (250 mL) and extracted once with chloroform (250 mL) .
  • the brine solution was continuously extracted and the product was crystallized as described above to afford an additional 178 g of crystalline product containing about 2% of thymine.
  • the combined yield was 1827 g (69.4%) .
  • HPLC indicated about 99.5% purity with the balance being the dimer.
  • Dimethoxytriphenylmethyl chloride (1765.7 g, 5.21 mol) was added as a solid in one portion.
  • the reaction was allowed to warm to -2°C over 1 h. (Note: The reaction was monitored closely by TLC (EtOAc) to determine when to stop the reaction so as to not generate the undesired bis-DMT substituted side product) .
  • the reaction was allowed to warm from -2 to 3°C over 25 min. then quenched by adding MeOH (300 mL) followed after 10 min by toluene (16 L) and water (16 L) .
  • the solution was transferred to a clear 50 L vessel with a bottom outlet, vigorously stirred for 1 minute, and the layers separated.
  • the aqueous layer was removed and the organic layer was washed successively with 10% aqueous citric acid (8 L) and water (12 L) .
  • the product was then extracted into the aqueous phase by washing the toluene solution with aqueous sodium hydroxide (0.5N, 16 L and 8 L) .
  • the combined aqueous layer was overlayed with toluene (12 L) and solid citric acid (8 moles, 1270 g) was added with vigorous stirring to lower the pH of the aqueous layer to 5.5 and extract the product into the toluene.
  • the organic layer was washed with water (10 L) and TLC of the organic layer indicated a trace of DMT-O-Me, bis DMT and dimer DMT.
  • the toluene solution was applied to a silica gel column (6 L sintered glass funnel containing approx. 2 kg of silica gel slurried with toluene (2 L) and TEA(25 mL) ) and the fractions were eluted with toluene (12 L) and EtOAc (3 x 4 L) using vacuum applied to a filter flask placed below the column.
  • the first EtOAc fraction containing both the desired product and impurities were resubjected to column chromatography as above.
  • the solution was co-evaporated with toluene (200 ml) at 50°C under reduced pressure, then cooled to room temperature and 2-cyanoethyl tetraisopropylphosphorodiamidite (900 g, 3.0 mol) and tetrazole (70 g, 1.0 mol) were added. The mixture was shaken until all tetrazole was dissolved, N-methylimidazole (20 ml) was added and the solution was left at room temperature for 5 hours. TEA (300 ml) was added, the mixture was diluted with DMF (3.5 L) and water (600 ml) and extracted with hexane (3 x 3L) .
  • the mixture was diluted with water (1.6 L) and extracted with the mixture of toluene (12 L) and hexanes (9 L) .
  • the upper layer was washed with DMF-water (7:3 v/v, 3x3 L) and water (3x3 L) .
  • the organic layer was dried (Na 2 S0 4 ) , filtered and evaporated.
  • the residue was co-evaporated with acetonitrile (2 x 2 L) under reduced pressure and dried in a vacuum oven (25°C, 0.1mm Hg, 40 h) to afford 1526 g of an off- white foamy solid (95%) .
  • Trimethylsilylchloride (1.60 L, 12.7 mol, 3.0 eq) was added over 30 min. while maintaining the internal temperature below -5°C, followed by a wash of anhydrous acetonitrile (1 L) .
  • the reaction was allowed to warm to 0°C and the reaction progress was confirmed by TLC (EtOAc, R f 0.68 and 0.87 for starting material and silyl product, respectively) .
  • triazole (2.34 kg, 33.8 mol, 8.0 eq) was added the reaction was cooled to -20 °C internal temperature (external -30°C).
  • reaction was cooled to -15°C and water (5 L) was slowly added at a rate to maintain the temperature below +10 °C in order to quench the reaction and to form a homogenous solution. (Caution: this reaction is initially very strongly exothermic) .
  • Approximately one-half of the reaction volume (22 L) was transferred by air pump to another vessel, diluted with EtOAc (12 L) and extracted with water (2 x 8 L) .
  • the second half of the reaction was treated in the same way.
  • the combined aqueous layers were back-extracted with EtOAc (8 L)
  • the organic layers were combined and concentrated in a 20 L rotary evaporator to an oily foam. The foam was coevaporated with anhydrous acetonitrile (4 L) to remove EtOAc.
  • 2 ' - (Dimethylaminooxyethoxy) nucleoside amidites (also known in the art as 2 ' -0- (dimethylaminooxyethyl) nucleoside amidites) are prepared as described in the following paragraphs.
  • Adenosine, cytidine and guanosine nucleoside amidites are prepared similarly to the thymidine (5- methyluridine) except the exocyclic amines are protected with a benzoyl moiety in the case of adenosine and cytidine and with isobutyryl in the case of guanosine.
  • the reaction vessel was cooled to ambient temperature and opened.
  • TLC EtOAc, R f 0.67 for desired product and R f 0.82 for ara-T side product
  • the solution was concentrated under reduced pressure (10 to lmm Hg) in a warm water bath (40-100°C) with the more extreme conditions used to remove the ethylene glycol .
  • the solution can be diluted with water and the product extracted into EtOAc) .
  • the residue was purified by column chromatography (2kg silica gel, EtOAc-hexanes gradient 1:1 to 4:1).
  • Triethylamine trihydrofluoride (3.91mL, 24.0mmol) was dissolved in dry THF and TEA (1.67mL, 12mmol, dry, stored over KOH) and added to 5 ' -0- tert-butyldiphenylsilyl-2 ' -0- [N,N- dimethylaminooxyethyl] -5-methyluridine (1.40g, 2.4mmol). The reaction was stirred at room temperature for 24 hrs and monitored by TLC (5% MeOH in CH 2 C1 2 ) .
  • the reaction mixture was stirred at ambient temperature for 4 h under inert atmosphere. The progress of the reaction was monitored by TLC (hexane : EtOAc 1:1) . The solvent was evaporated, then the residue was dissolved in EtOAc (70mL) and washed with 5% aqueous NaHC0 3 (40mL) . The EtOAc layer was dried over anhydrous Na 2 S0 4 , filtered, and concentrated.
  • 2 ' - (Aminooxyethoxy) nucleoside amidites (also known in the art as 2 ' -0- (aminooxyethyl) nucleoside amidites) are prepared as described in the following paragraphs. Adenosine, cytidine and thymidine nucleoside amidites are prepared similarly.
  • the 2 ' -0-aminooxyethyl guanosine analog may be obtained by selective 2 ' -O-alkylation of diaminopurine riboside.
  • Multigram quantities of diaminopurine riboside may be purchased from Schering AG (Berlin) to provide 2'-0-(2- ethylacetyl) diaminopurine riboside along with a minor amount of the 3'-0-isomer.
  • 2 ' -0- (2-ethylacetyl) diaminopurine riboside may be resolved and converted to 2'-0-(2- ethylacetyl) guanosine by treatment with adenosine deaminase .
  • the hydroxyl group may be displaced by N-hydroxyphthalimide via a Mitsunobu reaction, and the protected nucleoside may be phosphitylated as usual to yield 2-N-isobutyryl-6-0- diphenylcarbamoyl-2 ' -0- ( [2-phthalmidoxy] ethyl) -5 ' -0- (4,4 ' - dimethoxytrityl) guanosine-3 ' - [ (2-cyanoethyl) -N,N- diisopropylphosphoramidite] 2 ' -dimeth laminoethoxyethoxy (2'-DMAEOE) nucleoside amidites
  • 2 ' -dimethylaminoethoxyethoxy nucleoside amidites (also known in the art as 2 ' -O-dimethylaminoethoxyethyl , i.e., 2 ' -0- CH 2 -0-CH 2 -N(CH 2 ) 2 , or 2 ' -DMAEOE nucleoside amidites) are prepared as follows. Other nucleoside amidites are prepared similarly.
  • the crude solution was concentrated, the residue was diluted with water (200 mL) and extracted with hexanes (200 mL) .
  • the product was extracted from the aqueous layer with EtOAc (3 x 200 mL) and the combined organic layers were washed once with water, dried over anhydrous sodium sulfate, filtered and concentrated.
  • the residue was purified by silica gel column chromatography (eluted with 5:100:2 MeOH/CH 2 Cl 2 /TEA) as the eluent. The appropriate fractions were combined and evaporated to afford the product as a white solid.
  • Phosphinate oligonucleotides are prepared as described in U.S. Patent 5,508,270, herein incorporated by reference.
  • Alkyl phosphonate oligonucleotides are prepared as described in U.S. Patent 4,469,863, herein incorporated by reference .
  • 3' -Deoxy-3' -methylene phosphonate oligonucleotides are prepared as described in U.S. Patents 5,610,289 or 5,625,050, herein incorporated by reference.
  • Phosphoramidite oligonucleotides are prepared as described in U.S. Patent, 5,256,775 or U.S. Patent 5,366,878, herein incorporated by reference.
  • Alkylphosphonothioate oligonucleotides are prepared as described in published PCT applications PCT/US94/00902 and PCT/US93/06976 (published as WO 94/17093 and WO 94/02499, respectively), herein incorporated by reference.
  • 3 ' -Deoxy-3 ' -amino phosphoramidate oligonucleotides are prepared as described in U.S. Patent 5,476,925, herein incorporated by reference .
  • Phosphotriester oligonucleotides are prepared as described in U.S. Patent 5,023,243, herein incorporated by reference .
  • Borano phosphate oligonucleotides are prepared as described in U.S. Patents 5,130,302 and 5,177,198, both herein incorporated by reference.
  • Formacetal and thioformacetal linked oligonucleosides are prepared as described in U.S. Patents 5,264,562 and 5,264,564, herein incorporated by reference.
  • Ethylene oxide linked oligonucleosides are prepared as described in U.S. Patent 5,223,618, herein incorporated by reference.
  • PNAs Peptide nucleic acids
  • Nucleic Acids (PNA) : Synthesis, Properties and Potential Applications, Bioorganic & Medicinal Chemistry, 1996, 4, 5-23. They may also be prepared in accordance with U.S. Patents 5,539,082, 5,700,922, and 5,719,262, herein incorporated by reference.
  • Chimeric oligonucleotides, oligonucleosides or mixed oligonucleotides/oligonucleosides of the invention can be of several different types. These include a first type wherein the "gap" segment of linked nucleosides is positioned between 5' and 3' "wing" segments of linked nucleosides and a second "open end” type wherein the "gap” segment is located at either the 3' or the 5' terminus of the oligomeric compound. Oligonucleotides of the first type are also known in the art as “gapmers” or gapped oligonucleotides. Oligonucleotides of the second type are also known in the art as “hemimers” or "wingmers” .
  • Oligonucleotides are synthesized using the automated synthesizer and 2 ' -deoxy-5 ' - dimethoxytrityl-3 ' -O-phosphoramidite for the DNA portion and 5 ' -dimethoxytrityl-2 ' -O-methyl-3 ' -O-phosphoramidite for 5' and 3' wings.
  • the standard synthesis cycle is modified by incorporating coupling steps with increased reaction times for the 5 ' -dimethoxytrityl-2 ' -O-methyl-3 ' -O-phosphoramidite .
  • the fully protected oligonucleotide is cleaved from the support and deprotected in concentrated ammonia (NH 4 0H) for 12-16 hr at 55°C.
  • the deprotected oligo is then recovered by an appropriate method (precipitation, column chromatography, volume reduced in vacuo and analyzed spetrophotometrically for yield and for purity by capillary electrophoresis and by mass spectrometry.
  • chimeric oligonucleotides chimeric oligonucleosides and mixed chimeric oligonucleotides/oligonucleosides are synthesized according to United States patent 5,623,065, herein incorporated by reference .
  • Example 6 Oligonucleotide Isolation After cleavage from the controlled pore glass solid support and deblocking in concentrated ammonium hydroxide at 55°C for 12-16 hours, the oligonucleotides or oligonucleosides are recovered by precipitation out of 1 M NH 4 OAc with >3 volumes of ethanol. Synthesized oligonucleotides were analyzed by electrospray mass spectroscopy (molecular weight determination) and by capillary gel electrophoresis and judged to be at least 70% full length material.
  • Oligonucleotide Synthesis - 96 Well Plate Format Oligonucleotides were synthesized via solid phase P(III) phosphoramidite chemistry on an automated synthesizer capable of assembling 96 sequences simultaneously in a 96-well format. Phosphodiester internucleotide linkages were afforded by oxidation with aqueous iodine. Phosphorothioate internucleotide linkages were generated by sulfurization utilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide (Beaucage Reagent) in anhydrous acetonitrile.
  • Standard base-protected beta-cyanoethyl-diiso-propyl phosphoramidites were purchased from commercial vendors (e.g. PE-Applied Biosystems, Foster City, CA, or Pharmacia, Piscataway, NJ) .
  • Non-standard nucleosides are synthesized as per standard or patented methods. They are utilized as base protected beta- cyanoethyldiisopropyl phosphoramidites .
  • Oligonucleotides were cleaved from support and deprotected with concentrated NHOH at elevated temperature
  • the concentration of oligonucleotide in each well was assessed by dilution of samples and UV absorption spectroscopy.
  • the full-length integrity of the individual products was evaluated by capillary electrophoresis (CE) in either the 96-well format (Beckman P/ACETM MDQ) or, for individually prepared samples, on a commercial CE apparatus (e.g., Beckman P/ACETM 5000, ABI 270). Base and backbone composition was confirmed by mass analysis of the compounds utilizing electrospray-mass spectroscopy. All assay test plates were diluted from the master plate using single and multi-channel robotic pipettors. Plates were judged to be acceptable if at least 85% of the compounds on the plate were at least 85% full length.
  • the effect of antisense compounds on target nucleic acid expression can be tested in any of a variety of cell types provided that the target nucleic acid is present at measurable levels. This can be routinely determined using, for example, PCR or Northern blot analysis. The following cell types are provided for illustrative purposes, but other cell types can be routinely used, provided that the target is expressed in the cell type chosen. This can be readily determined by methods routine in the art, for example Northern blot analysis, ribonuclease protection assays, or RT-PCR.
  • T-24 cells The human transitional cell bladder carcinoma cell line
  • T-24 was obtained from the American Type Culture Collection (ATCC) (Manassas, VA) .
  • ATCC American Type Culture Collection
  • T-24 cells were routinely cultured in complete McCoy's 5A basal media (Invitrogen Corporation, Carlsbad, CA) supplemented with 10% fetal calf serum (Invitrogen Corporation, Carlsbad, CA) , penicillin 100 units per mL, and streptomycin 100 micrograms per mL (Invitrogen Corporation, Carlsbad, CA) .
  • Cells were routinely passaged by trypsinization and dilution when they reached 90% confluence.
  • Cells were seeded into 96-well plates (Falcon-Primaria #3872) at a density of 7000 cells/well for use in RT-PCR analysis.
  • cells may be seeded onto 100 mm or other standard tissue culture plates and treated similarly, using appropriate volumes of medium and oligonucleotide .
  • A549 cells The human lung carcinoma cell line A549 was obtained from the American Type Culture Collection (ATCC) (Manassas, VA) .
  • A549 cells were routinely cultured in DMEM basal media (Invitrogen Corporation, Carlsbad, CA) supplemented with 10% fetal calf serum (Invitrogen Corporation, Carlsbad, CA) , penicillin 100 units per mL, and streptomycin 100 micrograms per mL (Invitrogen Corporation, Carlsbad, CA) . Cells were routinely passaged by trypsinization and dilution when they reached 90% confluence.
  • ATCC American Type Culture Collection
  • NHDF Human neonatal dermal fibroblast
  • HEK cells Human embryonic keratinocytes (HEK) were obtained from the Clonetics Corporation (Walkersville, MD) . HEKs were routinely maintained in Keratinocyte Growth Medium (Clonetics Corporation, Walkersville, MD) formulated as recommended by the supplier. Cells were routinely maintained for up to 10 passages as recommended by the supplier.
  • oligonucleotide When cells reached 70% confluency, they were treated with oligonucleotide. For cells grown in 96-well plates, wells were washed once with 100 ⁇ L 0PTI-MEMTM-1 reduced-serum medium (Invitrogen Corporation, Carlsbad, CA) and then treated with 130 ⁇ L of OPTI-MEMTM-l containing 3.75 ⁇ g/mL LIPOFECTINTM (Invitrogen Corporation, Carlsbad, CA) and the desired concentration of oligonucleotide. After 4-7 hours of treatment, the medium was replaced with fresh medium. Cells were harvested 16-24 hours after oligonucleotide treatment. The concentration of oligonucleotide used varies from cell line to cell line.
  • the cells are treated with a positive control oligonucleotide at a range of concentrations.
  • a positive control oligonucleotide is selected from either ISIS 13920 (TCCGTCATCGCTCCTCAGGG, SEQ ID NO: 1) which is targeted to human H-ras, or ISIS 18078, (GTGCGCGCGAGCCCGAAATC, SEQ ID NO:
  • Jun-N-terminal kinase-2 JNK2
  • Both controls are 2 ' -O-methoxyethyl gapmers (2 ' -0- methoxyethyls shown in bold) with a phosphorothioate backbone.
  • the positive control oligonucleotide is ISIS 15770, ATGCATTCTGCCCCCAAGGA, SEQ ID NO : 3, a 2 ' -0- methoxyethyl gapmer (2 ' -0-methoxyethyls shown in bold) with a phosphorothioate backbone which is targeted to both mouse and rat c-raf .
  • the concentration of positive control oligonucleotide that results in 80% inhibition of c-Ha-ras (for ISIS 13920) or c-raf (for ISIS 15770) mRNA is then utilized as the screening concentration for new oligonucleotides in subsequent experiments for that cell line. If 80% inhibition is not achieved, the lowest concentration of positive control oligonucleotide that results in 60% inhibition of H-ras or c-raf mRNA is then utilized as the oligonucleotide screening concentration in subsequent experiments for that cell line. If 60% inhibition is not achieved, that particular cell line is deemed as unsuitable for oligonucleotide transfection experiments.
  • concentrations of antisense oligonucleotides used herein are from 50 nM to 300 nM.
  • G protein-coupled receptor kinase 6 expression can be assayed in a variety of ways known in the art.
  • G protein-coupled receptor kinase 6 mRNA levels can be quantitated by, e.g., Northern blot analysis, competitive polymerase chain reaction (PCR) , or real-time PCR (RT-PCR) . Real-time quantitative PCR is presently preferred.
  • RNA analysis can be performed on total cellular RNA or poly (A) + mRNA. The preferred method of RNA analysis of the present invention is the use of total cellular RNA as described in other examples herein. Methods of RNA isolation are taught in, for example, Ausubel, F.M. et al .
  • Protein levels of G protein-coupled receptor kinase 6 can be quantitated in a variety of ways well known in the art, such as immunoprecipitation, Western blot analysis (immunoblotting) , ELISA or fluorescence-activated cell sorting (FACS) .
  • Antibodies directed to G protein-coupled receptor kinase 6 can be identified and obtained from a variety of sources, such as the MSRS catalog of antibodies (Aerie Corporation, Birmingham, MI) , or can be prepared via conventional antibody generation methods . Methods for preparation of polyclonal antisera are taught in, for example, Ausubel, F.M. et al . , ( Current Protocols in Molecular Biology, Volume 2, pp.
  • Poly (A) + mRNA was isolated according to Miura et al . , ( Clin . Chem . , 1996, 42 , 1758-1764). Other methods for poly (A) + mRNA isolation are taught in, for example, Ausubel, F.M. et al . , ( Current Protocols in Molecular Biology, Volume 1, pp. 4.5.1-4.5.3, John Wiley & Sons, Inc., 1993). Briefly, for cells grown on 96-well plates, growth medium was removed from the cells and each well was washed with 200 ⁇ L cold PBS.
  • lysis buffer (10 mM Tris-HCl, pH 7.6 , 1 mM EDTA, 0.5 M NaCl, 0.5% NP-40, 20 M vanadyl-ribonucleoside complex) was added to each well, the plate was gently agitated and then incubated at room temperature for five minutes . 55 ⁇ L of lysate was transferred to Oligo d(T) coated 96-well plates (AGCT Inc., Irvine CA) . Plates were incubated for 60 minutes at room temperature, washed 3 times with 200 ⁇ L of wash buffer (10 mM Tris-HCl pH 7.6 , 1 mM EDTA, 0.3 M NaCl) .
  • the plate was blotted on paper towels to remove excess wash buffer and then air-dried for 5 minutes.
  • 60 ⁇ L of elution buffer (5 mM Tris-HCl pH 7.6), preheated to 70°C, was added to each well, the plate was incubated on a 90°C hot plate for 5 minutes, and the eluate was then transferred to a fresh 96-well plate.
  • Cells grown on 100 mm or other standard plates may be treated similarly, using appropriate volumes of all solutions.
  • the repetitive pipetting and elution steps may be automated using a QIAGEN Bio-Robot 9604 (Qiagen, Inc., Valencia CA) . Essentially, after lysing of the cells on the culture plate, the plate is transferred to the robot deck where the pipetting, DNase treatment and elution steps are carried out .
  • Quantitation of G protein-coupled receptor kinase 6 mRNA levels was determined by real-time quantitative PCR using the ABI PRISMTM 7700 Sequence Detection System (PE-Applied Biosystems, Foster City, CA) according to manufacturer's instructions. This is a closed-tube, non-gel-based, fluorescence detection system which allows high-throughput quantitation of polymerase chain reaction (PCR) products in real-time. As opposed to standard PCR in which amplification products are quantitated after the PCR is completed, products in real-time quantitative PCR are quantitated as they accumulate. This is accomplished by including in the PCR reaction an oligonucleotide probe that anneals specifically between the forward and reverse PCR primers, and contains two fluorescent dyes.
  • PCR polymerase chain reaction
  • a reporter dye e.g., FAM or JOE, obtained from either PE-Applied Biosystems, Foster City, CA, Operon Technologies Inc., Alameda, CA or Integrated DNA Technologies Inc., Coralville, IA
  • a quencher dye e.g., TAMRA, obtained from either PE- Applied Biosystems, Foster City, CA, Operon Technologies Inc., Alameda, CA or Integrated DNA Technologies Inc., Coralville, IA
  • TAMRA obtained from either PE- Applied Biosystems, Foster City, CA, Operon Technologies Inc., Alameda, CA or Integrated DNA Technologies Inc., Coralville, IA
  • annealing of the probe to the target sequence creates a substrate that can be cleaved by the 5 ' -exonuclease activity of Taq polymerase.
  • cleavage of the probe by Taq polymerase releases the reporter dye from the remainder of the probe (and hence from the quencher moiety) and a sequence-specific fluorescent signal is generated.
  • additional reporter dye molecules are cleaved from their respective probes, and the fluorescence intensity is monitored at regular intervals by laser optics built into the ABI PRISMTM 7700 Sequence Detection System.
  • a series of parallel reactions containing serial dilutions of mRNA from untreated control samples generates a standard curve that is used to quantitate the percent inhibition after antisense oligonucleotide treatment of test samples.
  • primer-probe sets specific to the target gene being measured are evaluated for their ability to be "multiplexed" with a GAPDH amplification reaction.
  • multiplexing both the target gene and the internal standard gene GAPDH are amplified concurrently in a single sample.
  • mRNA isolated from untreated cells is serially diluted. Each dilution is amplified in the presence of primer-probe sets specific for GAPDH only, target gene only ( "single-plexing" ) , or both (multiplexing).
  • standard curves of GAPDH and target mRNA signal as a function of dilution are generated from both the single-plexed and multiplexed samples.
  • PCR reagents were obtained from Invitrogen Corporation, (Carlsbad, CA) .
  • RT-PCR reactions were carried out by adding 20 ⁇ L PCR cocktail (2.5x PCR buffer (-MgCl2) , 6.6 mM MgCl2, 375 ⁇ M each of dATP, dCTP, dCTP and dGTP, 375 nM each of forward primer and reverse primer, 125 nM of probe, 4 Units
  • RNAse inhibitor 1.25 Units PLATINUM® Taq, 5 Units MuLV reverse transcriptase, and 2.5x ROX dye
  • the RT reaction was carried out by incubation for 30 minutes at 48°C. Following a 10 minute incubation at 95°C to activate the PLATINUM® Taq, 40 cycles of a two-step PCR protocol were carried out: 95°C for 15 seconds (denaturation) followed by 60°C for 1.5 minutes (annealing/extension) .
  • Gene target quantities obtained by real time RT-PCR are normalized using either the expression level of GAPDH, a gene whose expression is constant, or by quantifying total RNA using RiboGreenTM (Molecular Probes, Inc. Eugene, OR).
  • GAPDH expression is quantified by real time RT-PCR, by being run simultaneously with the target, multiplexing, or separately.
  • Total RNA is quantified using RiboGreenTM RNA quantification reagent from Molecular Probes. Methods of RNA quantification by RiboGreenTM are taught in Jones, L.J., et al , (Analytical Biochemistry, 1998, 265, 368-374) .
  • RiboGreenTM working reagent 170 ⁇ L of RiboGreenTM working reagent (RiboGreenTM reagent diluted 1:350 in lOmM Tris-HCl, 1 mM EDTA, pH 7.5) is pipetted into a 96-well plate containing 30 ⁇ L purified, cellular RNA. The plate is read in a CytoFluor . 4000 (PE Applied Biosystems) with excitation at 480nm and emission at 520nm.
  • Probes and primers to human G protein-coupled receptor kinase 6 were designed to hybridize to a human G protein- coupled receptor kinase 6 sequence, using published sequence information (GenBank accession number NM__002082.1, incorporated herein as SEQ ID NO: 4) .
  • the PCR primers were: forward primer: CCCTGGTCAACCCTCAAACA (SEQ ID NO: 5) reverse primer: CGGATTATTGCTGGGCACAT (SEQ ID NO : 6) and the PCR probe was: FAM-TCCGGACTCCCCTCATAACAATAGAC-TAMRA (SEQ ID NO: 7) where FAM is the fluorescent dye and TAMRA is the quencher dye .
  • PCR primers were : forward primer: GAAGGTGAAGGTCGGAGTC (SEQ ID NO: 8) reverse primer: GAAGATGGTGATGGGATTTC (SEQ ID NO: 9) and the PCR probe was: 5' JOE-CAAGCTTCCCGTTCTCAGCC- TAMRA 3' (SEQ ID NO: 10) where JOE is the fluorescent reporter dye and TAMRA is the quencher dye .
  • RNAZOLTM TEL-TEST "B” Inc., Friendswood, TX
  • Total RNA was prepared following manufacturer's recommended protocols. Twenty micrograms of total RNA was fractionated by electrophoresis through 1.2% agarose gels containing 1.1% formaldehyde using a MOPS buffer system (AMRESCO, Inc. Solon, OH) .
  • QUICKHYBTM hybridization solution (Stratagene, La Jolla, CA) using manufacturer's recommendations for stringent conditions.
  • a human G protein-coupled receptor kinase 6 specific probe was prepared by PCR using the forward primer CCCTGGTCAACCCTCAAACA
  • Hybridized membranes were visualized and quantitated using a PHOSPHORIMAGERTM and IMAGEQUANTTM Software V3.3
  • oligonucleotides were designed to target different regions of the human G protein-coupled receptor kinase 6 RNA, using published sequences (GenBank accession number NM_002082.1, incorporated herein as SEQ ID NO : 4) .
  • the oligonucleotides are shown in Table 1. "Target site” indicates the first (5'- most) nucleotide number on the particular target sequence to which the oligonucleotide binds.
  • All compounds in Table 1 are chimeric oligonucleotides ("gapmers") 20 nucleotides in length, composed of a central "gap" region consisting of ten 2 ' -deoxynucleotides, which is flanked on both sides (5' and 3' directions) by five-nucleotide "wings" .
  • the wings are composed of 2 ' -methoxyethyl (2 ' -MOE) nucleotides .
  • the compounds were analyzed for their effect on human G protein-coupled receptor kinase 6 mRNA levels by quantitative real-time PCR as described in other examples herein. Data are averages from two experiments in which A549 cells were treated with the oligonucleotides of the present invention.
  • the positive control for each datapoint is identified in the table by sequence ID number. If present, "N.D.” indicates "no data”.
  • SEQ ID NOs 12, 13, 14, 15, 16, 17, 21, 23, 26, 28, 30, 31, 32, 33, 36, 38, 39, 41, 42, 43, 44, 45, 46, 47, 48, 50, 52, 53, 55, 64, 65, 66, 67, 68, 69, 70, 71, 76, 77, 78, 79, 80, 81, 82, 85 and 87 demonstrated at least 40% inhibition of human G protein-coupled receptor kinase 6 expression in this assay and are therefore preferred.
  • the target sites to which these preferred sequences are complementary are herein referred to as "preferred target regions" and are therefore preferred sites for targeting by compounds of the present invention. These preferred target regions are shown in Table 2.
  • Target site indicates the first (5 '-most) nucleotide number of the corresponding target nucleic acid. Also shown in Table 2 is the species in which each of the preferred target regions was found.

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Abstract

La présente invention a trait à des composés, des compositions et des procédés antisens permettant la modulation de l'expression de la protéine kinase GR 6. Les compositions comportent des composés antisens, notamment des oligonucléotides antisens, ciblés vers des acides nucléiques codant pour la protéine kinase GR 6. L'invention a trait également à des procédés d'utilisation desdits composés pour la modulation de l'expression de la protéine kinase GR 6 et pour le traitement de maladies liées à l'expression de la protéine kinase GR 6.
PCT/US2003/017174 2002-05-31 2003-05-29 Modulation antisens de l'expression de la proteine kinase gr 6 WO2003104397A2 (fr)

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US20040076621A1 (en) * 2002-10-16 2004-04-22 Isis Pharmaceuticals Inc. Antisense modulation of CD36 expression
US20100152280A1 (en) * 2004-05-24 2010-06-17 Isis Pharmaceuticals, Inc. Modulation of sid-1 expression
EP2687609B1 (fr) 2008-11-10 2017-01-04 The United States of America, as represented by The Secretary, Department of Health and Human Services Méthode de traitement de tumeurs solides
WO2010093872A2 (fr) 2009-02-13 2010-08-19 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Procédé moléculaire de diagnostic et de pronostic du cancer
WO2011163466A1 (fr) 2010-06-23 2011-12-29 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Régulation de la pigmentation cutanée par la neuroréguline-1 (nrg-1)
US9150926B2 (en) 2010-12-06 2015-10-06 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Diagnosis and treatment of adrenocortical tumors using human microRNA-483
WO2014197680A1 (fr) 2013-06-05 2014-12-11 Salk Institute For Biological Studies Agonistes du récepteur de la vitamine d pour le traitement de maladies impliquant l'activité de cxcl12
EP3055426B1 (fr) 2013-10-09 2019-06-19 The United States of America as represented by The Secretary Department of Health and Human Services Détection du virus de l'hépatite delta (vhd) pour le diagnostic et le traitement du syndrome de sjögren et du lymphome
WO2015074057A1 (fr) 2013-11-18 2015-05-21 University Of Pittsburgh - Of The Commonwealth System Of Higher Education Administration à base de microsphères et manipulation ex vivo de cellules dendritiques pour thérapies auto-immunes
WO2022187440A1 (fr) 2021-03-03 2022-09-09 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Proprotéine la en tant que nouveau régulateur de l'ostéoclastogenèse
CA3235862A1 (fr) 2021-10-22 2023-04-27 Takahiro Ochiya Procedes de fabrication de vesicules extracellulaires, et compositions et procedes d'utilisation de celles-ci
WO2024036315A1 (fr) 2022-08-12 2024-02-15 Endorel Biosciences Llc Agent d'administration à base de peptide et son procédé de fabrication et d'utilisation

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